Optoelectronic detection system

ABSTRACT

The invention described herein provides methods for the detection of soluble antigens. In particular, the methods provide for the detection of soluble proteins and chemicals. In addition, the invention provides methods of detecting a nucleic acid sequence in a sample. Also described is an emittor cell comprising an Fc receptor and an emittor molecule for the detection of a target particle in a sample wherein the target particle to be detected is bound by one or more antibodies. Also provided is an optoelectronic sensor device for detecting a target particle in a plurality of samples.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.10/467,242, filed Jan. 16, 2004, which is the U.S. National stage ofInternational Application No. PCT/US02/03606, filed Feb. 6, 2002,published in English, which claims the benefit of U.S. ProvisionalApplication No. 60/266,977, filed Feb. 7, 2001.

The entire teachings of the above applications are incorporated hereinby reference.

GOVERNMENT SUPPORT

This invention was made with Government funds from U.S. Air Forcecontract no. F19628-00-C-0002. The Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

The need for small, fast, and sensitive detectors of biological agentswhich are able to monitor an environment for extended periods of time isunderscored by the proliferation of biological and chemical weapons, thepoor man's nuclear weapon. Under battlefield conditions, a usefuldetector would rapidly alert a soldier when a specific biological orchemical agent is detected so that countermeasures can quickly beimplemented.

Such detectors would be useful in non-military applications as well.Rapid detection of antibiotic-resistant bacteria in a patient would helpclinicians select a more effective therapeutic regimen. Continuousmonitoring of a city's drinking water supply would provide early warningof potential pathogens, giving public works officials more time tomanage the potential health risks to the public. In addition, the use ofthese detectors in meat and poultry inspections would be a significantimprovement over the current “poke-and-smell” procedure. In general,such detectors are sorely needed analytical and diagnostic applicationswithin the fields of medicine (e.g., veterinary medicine), agriculture,environmental protection (e.g., to diagnose sick building syndrome), andfood processing or regulation.

All vertebrates acquire a specific immune response to a foreign agent(antigen) in part by generating an immense diversity of antibodymolecules. Antibody molecules bind to antigen with high specificity,e.g., they can differentially bind to two closely related strains ofbacteria, viruses, protein, nucleic acid, fungus, protozoa,multicellular parasite, or prion, as well as products produced orinduced by those particles.

Antibodies are produced by B cells, a crucial component of the immunesystem. An antigen can activate a B cell by binding to antibodies on itssurface, leading to a cascade of intracellular biochemical reactionswhich causes a calcium ion influx into the cytosol of the B cell.

For a review of antibody structure and function and B cell activation,see Paul, editor, Fundamental Immunology, 3rd ed., Raven Press, New York(1993).

Devices that exploit antibody diversity for detection of multiple andrare target particles or antigens have been described in U.S. Pat. No.6,087,114 and U.S. Pat. No. 6,248,542.

These devices generally include a liquid medium containing sensor cells(e.g., a B cell, macrophage or fibroblast), also referred to herein as“CANARY” cells or emittor cells, an optical detector, and the liquidmedium receiving target particles to be detected. Each of the cells hasreceptors (e.g., chimeric or single chain antibodies) which areexpressed on its surface and are specific for the antigen to bedetected. Binding of the antigen to the receptor results in a signalingpathway involving chemical or biochemical changes (e.g., an increase incalcium concentration). The cells also contain emitter molecules (e.g.,aequorin or indo-1) in their cytosol which can emit photons in responseto the signaling pathway (e.g., increased calcium concentration in thecytosol). The detector can be separated from the medium containing thecells by a covering (e.g., glass) that is transparent to the photons.Such a covering can serve to support the medium, protect a fragilesurface of the detector, or be used as a lens. The optical detector,e.g., a charge-coupled device (CCD) is able to detect the photonsemitted from the cells in response to the receptor-mediated signalingpathway and indicate to the user that the antigen to be detected ispresent. Other optical detectors which can be used in the device includephotomultiplier tubes, photodiodes, complimentary metal oxidesemiconductor (CMOS) imagers, avalanche photodiodes, andimage-intensified charge-coupled devices (ICCD) (see for example, thoseavailable from Photek Ltd., East Sussex, UK). In some embodiments, theoptical detector is able to distinguish individual cells.

SUMMARY OF THE INVENTION

The invention described herein modifies and advances the devicesdescribed in U.S. Pat. No. 6,087,114 and U.S. Pat. No. 6,248,542 toprovide methods for the detection of soluble antigens. In particular,the methods provide for the detection of soluble proteins and chemicals.In addition, the invention described herein provides methods ofdetecting a nucleic acid sequence in a sample. Furthermore, alsoprovided herein is an emittor cell comprising an Fc receptor and anemittor molecule for the detection of a target particle in a samplewherein the target particle to be detected is bound by one or moreantibodies. Also provided is an optoelectronic sensor device fordetecting a target particle in a plurality of samples using a photondetector.

Detection of a target particle (such as a soluble antigen or a nucleicacid) is mediated in part by binding of the target particle to areceptor, either directly or indirectly, expressed on the cell surfaceof an emittor cell. Direct binding can be via a receptor, such as anantibody, which binds directly and specifically to the target particle.Indirect binding of the target particle can be through an Fc receptorthat binds to an antibody that has been attached (bound) to the targetparticle.

In one embodiment of the invention, provided herein is a method ofdetecting a nucleic acid sequence in a sample comprising the steps of a)combining the sample with at least one antigen-conjugatedoligonucleotide under conditions suitable for hybridization of theantigen-conjugated oligonucleotide to the nucleic acid sequence; b)adding an emittor cell, wherein said emittor cell comprises one or morereceptors which bind to the antigen of the antigen-conjugatedoligonucleotides, wherein binding of the one or more receptors to theantigen results in an increase in intracellular calcium, and an emittormolecule that emits a photon in response to the increase inintracellular calcium; and c) measuring photon emission from the emittorcell, thereby detecting a nucleic acid sequence in a sample.

In another embodiment of the invention, provided herein is a method ofdetecting a nucleic acid sequence in a sample comprising the steps of a)combining the sample with a plurality of antigen-conjugatedoligonucleotides under conditions suitable for hybridization of theantigen-conjugated oligonucleotides to the nucleic acid sequence; b)adding an emittor cell, wherein said emittor cell comprises one or morereceptors which binds to the antigen of the antigen-conjugatedoligonucleotides, wherein binding of the one or more receptors to theantigen results in an increase in intracellular calcium, and an emittormolecule that emits a photon in response to the increase inintracellular calcium; and c) measuring photon emission from the emittorcell, thereby detecting a nucleic acid sequence in a sample.

In a further embodiment of the invention, provided herein is a method ofdetecting a nucleic acid sequence in a sample comprising the steps of a)combining i) the sample being tested; ii) at least oneantigen-conjugated oligonucleotide that is complementary to the nucleicacid sequence; and iii) a solid substrate comprising at least oneoligonucleotide complementary to the nucleic acid sequence bound to thesolid substrate; under conditions suitable for hybridization of theantigen-conjugated oligonucleotide and the oligonucleotide bound to thesolid substrate for hybridizing to the nucleic acid sequence, therebyproducing a solid substrate comprising the nucleic acid sequence havingat least one hybridized antigen-conjugated oligonucleotide; b) adding tothe solid substrate comprising the nucleic acid sequence having at leastone hybridized antigen-conjugated oligonucleotide an emittor cellcomprising one or more receptors which binds to the antigen of theantigen-conjugated oligonucleotide, wherein binding of the one or morereceptors to the antigen results in an increase in intracellularcalcium, and wherein said emittor cell further comprises an emittormolecule that emits a photon in response to the increase inintracellular calcium; and c) measuring photon emission from the emittorcell, thereby detecting a nucleic acid sequence in a sample.

In another embodiment of the invention, provided herein is a method ofdetecting a nucleic acid sequence in a sample comprising the steps of a)combining the sample with at least one antigen-conjugatedoligonucleotide under conditions suitable for hybridization of theantigen-conjugated oligonucleotide to the nucleic acid sequence therebyproducing an antigen-conjugated hybridization complex; b) adding one ormore antibodies specific for the antigen of the antigen-conjugatedhybridization complex; c) adding an emittor cell comprising an Fcreceptor, wherein binding of the Fc receptor to the one or moreantibodies results in an increase in intracellular calcium, and whereinsaid emittor cell further comprises an emittor molecule that emits aphoton in response to the increase in intracellular calcium; and d)measuring photon emission from the emittor cell, thereby detecting atarget particle in a sample.

In another embodiment of the invention, provided herein is a method ofdetecting a target particle in a sample comprising the steps of a)combining the sample with i) an antibody specific for the targetparticle; and ii) an emittor cell comprising an Fc receptor, whereinbinding of the Fc receptor to the antibody results in an increase inintracellular calcium, and wherein said emittor cell further comprisesan emittor molecule that emits a photon in response to the increase inintracellular calcium; and b) measuring photon emission from the emittorcell, thereby detecting a target particle in a sample

In a further embodiment, provided herein is a method of detecting asoluble antigen in a sample comprising the steps of a) combining thesample with i) one or more antibodies that bind to two differentepitopes on the soluble antigen; and ii) an emittor cell comprising anFc receptor, wherein binding of the Fc receptor to the one or moreantibodies results in an increase in intracellular calcium, and whereinsaid emittor cell further comprises an emittor molecule that emits aphoton in response to the increase in intracellular calcium; and b)measuring photon emission from the emittor cell, thereby detecting atarget particle in a sample.

Also provided herein is an optoelectronic sensor device for detecting atarget particle in a plurality of samples using a photon detectorcomprising a) a rotor comprising a plurality of positions to hold aplurality of samples; b) one or more samples comprising a mixture of i)a sample to be tested for the target particle; and ii) a cell comprisinga receptor for the target particle, wherein binding of the receptor tothe target particle results in an increase in intracellular calcium, andwherein said cell further comprises an emittor molecule that emits aphoton in response to the increase in intracellular calcium; and c) aphoton detector located at a position to detect photons emitted from oneor more samples upon rotation of the rotor.

In a further embodiment of the invention, provided herein is anoptoelectronic sensor device for detecting a target particle in one ormore samples using a photon detector comprising a) a rotor comprising aplurality of positions to hold a plurality of samples; b) one or moresamples comprising a mixture of i) a sample to be tested for the targetparticle; ii) an antibody specific for the target particle; and ii) anemittor cell, wherein said emittor cell comprises an Fc receptor,wherein binding of the Fc receptor to the antibody results in anincrease in intracellular calcium, and wherein said emittor cell furthercomprises an emittor molecule that emits a photon in response to theincrease in intracellular calcium; and c) a photon detector located at aposition to detect photons emitted from one or more samples uponrotation of the rotor.

In another embodiment of the invention, provided herein is anoptoelectronic sensor device for detecting a nucleic acid sequence inone or more samples using a photon detector comprising a) a rotorcomprising a plurality of positions to hold a plurality of samples; b)one or more samples comprising a mixture of i) a sample to be tested forthe nucleic acid sequence; ii) at least one antigen-conjugatedoligonucleotide hybridized to the nucleic acid sequence; and iii) anemittor cell comprising one or more receptors which binds to the antigenof the antigen-conjugated oligonucleotides hybridized to the nucleicacid sequence, wherein binding of the one or more receptors to theantigen results in an increase in intracellular calcium, and whereinsaid emittor cell further comprises an emittor molecule that emits aphoton in response to the increase in intracellular calcium; and c) aphoton detector located at a position to detect photons emitted from oneor more samples upon rotation of the rotor.

In one embodiment, the rotor comprises sixteen positions to hold sixteensamples.

Furthermore, in another embodiment of the invention, provided herein isa method of detecting a soluble antigen in a sample comprising a)combining the sample with an emittor cell comprising one or moreantibodies that bind to two different epitopes on the soluble antigen,wherein binding of the one or more antibodies to the soluble antigenresults in an increase in intracellular calcium, and wherein saidemittor cell further comprises an emittor molecule that emits a photonin response to the increase in intracellular calcium; and b) measuringphoton emission from the emittor cell, thereby detecting a solubleantigen in a sample.

In another embodiment of the invention, provided herein is a method ofdetecting a soluble antigen in a sample comprising the steps of a)crosslinking the soluble antigen, thereby producing a crosslinkedantigen; b) combining with the crosslinked antigen with an emittor cellcomprising an antibody that binds to the crosslinked antigen, whereinbinding of the antibody to the crosslinked antigen results in anincrease in intracellular calcium, and wherein said emittor cell furthercomprises an emittor molecule that emits a photon in response to theincrease in intracellular calcium; and c) measuring photon emission fromthe emittor cell, thereby detecting a soluble antigen in a sample.

In a further embodiment of the invention, provided herein is a method ofdetecting a soluble antigen in a sample comprising the steps of a)crosslinking the soluble antigen to a solid substrate, thereby producinga crosslinked soluble antigen bound to a solid substrate; b) adding anemittor cell to the crosslinked soluble antigen bound to the solidsubstrate, wherein said emittor cell comprises an antibody that binds anepitope on the soluble antigen, wherein binding of the antibody to thecrosslinked soluble antigen bound to the solid support results in anincrease in intracellular calcium, and wherein said emittor cell furthercomprises an emittor molecule that emits a photon in response to theincrease in intracellular calcium; and c) measuring photon emission fromthe emittor cell, thereby detecting a soluble antigen in a sample.

In still another embodiment of the invention, provided herein is amethod of detecting a soluble antigen in a sample comprising the stepsof a) combining the sample with a solid substrate comprising a firstantibody that binds a first epitope on the soluble antigen, therebyproducing a crosslinked soluble antigen bound to a solid substrate; b)adding an emittor cell to the crosslinked soluble antigen bound to thesolid substrate, wherein said emittor cell comprises a second antibodythat binds a second epitope on the soluble antigen, wherein binding ofthe second antibody to the crosslinked soluble antigen bound to thesolid support results in an increase in intracellular calcium, andwherein said emittor cell further comprises an emittor molecule thatemits a photon in response to the increase in intracellular calcium; andc) measuring photon emission from the emittor cell, thereby detecting asoluble antigen in a sample.

In another embodiment of the invention, provided herein is a method ofdetecting a chemical in a sample comprising the steps of a) combiningthe chemical with a peptide, thereby producing a chemical-peptidecomplex; b) adding an emittor cell comprising one or more antibodiesthat bind to two different epitopes on the chemical-peptide complex,wherein binding of the one or more antibodies to the chemical-peptidecomplex results in an increase in intracellular calcium, and whereinsaid emittor cell further comprises an emittor molecule that emits aphoton in response to the increase in intracellular calcium; and c)measuring photon emission from the emittor cell, thereby detecting achemical in a sample.

In further embodiment of the invention, provided herein is a method ofdetecting a chemical in a sample comprising the steps of a) combiningthe chemical with a peptide, thereby producing a chemical-peptidecomplex; b) crosslinking the chemical-peptide complex, thereby producinga crosslinked chemical-peptide complex; c) combining with thecrosslinked chemical-peptide complex with an emittor cell comprising anantibody that binds to the crosslinked chemical-peptide complex, whereinbinding of the antibody to the crosslinked chemical-peptide complexresults in an increase in intracellular calcium, and wherein saidemittor cell further comprises an emittor molecule that emits a photonin response to the increase in intracellular calcium; and d) measuringphoton emission from the emittor cell, thereby detecting a solubleantigen in a sample.

In an additional embodiment of the invention, provided herein is amethod of detecting a chemical in a sample comprising the steps of a)combining the chemical with an antigen-conjugated peptide, therebyproducing a chemical-antigen-conjugated peptide complex; b) adding anemittor cell comprising one or more antibodies that bind to twodifferent epitopes on the chemical-antigen-conjugated peptide complex,wherein binding of the one or more antibodies to thechemical-antigen-conjugated peptide complex results in an increase inintracellular calcium, and wherein said emittor cell further comprisesan emittor molecule that emits a photon in response to the increase inintracellular calcium; and c) measuring photon emission from the emittorcell, thereby detecting a chemical in a sample.

In a further embodiment of the invention, provided herein is a method ofdetecting a chemical in a sample comprising the steps of a) combiningthe chemical with an antigen-conjugated peptide, thereby producing achemical-antigen-conjugated peptide complex; b) crosslinking thechemical-antigen-conjugated peptide complex, thereby producing acrosslinked chemical-antigen-conjugated peptide complex; c) adding anemittor cell comprising an antibody that binds to the crosslinkedchemical-antigen-conjugated peptide complex, wherein binding of theantibody to the chemical-antigen-conjugated peptide complex results inan increase in intracellular calcium, and wherein said emittor cellfurther comprises an emittor molecule that emits a photon in response tothe increase in intracellular calcium; and d) measuring photon emissionfrom the emittor cell, thereby detecting a soluble antigen in a sample.

The systems of the invention are useful in analytical and diagnosticapplications within the fields of medicine (e.g., veterinary medicine),agriculture, environmental protection (e.g., to diagnose sick buildingsyndrome), and food processing or regulation.

The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a schematic of the optoelectronic sensor cellular concept.

FIG. 2 is a schematic showing the general architecture of anoptoelectronic sensor having a sampler (trigger) for preliminary sensingof suspect agents.

FIG. 3 is a schematic illustrating the creation of cell lines for use inthe optoelectronic sensor.

FIG. 4 is a schematic of an integrated biological aerosol warning sensor(BAWS)/optoelectronic sensor system.

FIG. 5 illustrates the B cell response to foot-and-mouth disease virusin the optoelectronic sensor.

FIG. 6 illustrates a dry-impactor module for the optoelectronic sensor.

FIG. 7 is a schematic illustrating the effect of localization andmixing.

FIG. 8 illustrates the effect of localization using tularemia cells.

FIG. 9 illustrates an automated cell-delivery module for theoptoelectronic sensor.

FIG. 10 illustrates a dose response relationship for a sample oftularemia cells using the optoelectronic sensor.

FIG. 11 illustrate B cell resistance to chemical and biologicalcontamination.

FIG. 12 illustrates an automated centrifuge module for theoptoelectronic sensor.

FIG. 13 is a schematic illustrating an air impactor/optoelectronicsensor.

FIG. 14 is a schematic illustrating an optoelectronic sensor.

FIG. 15 illustrates an optics-photomultiplier (PMT) module for theoptoelectronic sensor.

FIG. 16 is a schematic illustrating an air impactor/optoelectronicsensor.

FIG. 17 is a schematic illustrating a multi-channel centrifuge in theoptoelectronic sensor.

FIG. 18 is a schematic illustrating a wet centrifuge/impactor concept inthe optoelectronic sensor.

FIG. 19 is a schematic illustrating a wet centrifuge/impactor concept inthe optoelectronic sensor.

FIG. 20 is a schematic of a custom tube for the optoelectronic sensor.

FIG. 21 illustrates an integrated dry-impactor/optoelectronic sensor.

FIG. 22 illustrates the effect of cell treatments on the response ofYersenia pestis specific B cells.

FIG. 23 illustrates an impactor configured to collect aerosol samples.

FIG. 24 is a schematic overview of the concept underlying the “CANARY”sensor. B cells have been modified such that they express aequorinwithin the cell interior and antibodies to pathogen on the cell surface.In the presence of pathogen, the antibodies are “crosslinked”(immobilized, aggregated) on the surface of the cell, stimulating asignaling cascade that results in increased intracellular calcium.Aequorin responds to this increase in intracellular calcium by oxidizingaequorin, and emitting light. Photon output can be monitored using aPMT.

FIG. 25 is a schematic of DNA detection. Oligonucleotides complementaryto target DNA sequences and containing a terminal digoxigenin label arehybridized to the target DNA. Multiple digoxigenin-labeledoligonucleotides bound along the target DNA bind to digoxigeninantibodies on the surface of the CANARY cell (emittor cell), stimulatinglight emission.

FIGS. 26A-C are graphs. CANARY cells (emittor cells) expressingantibodies against digoxigenin can be stimulated by digoxigenin-labeledDNA. Emittor cells expressing antibody against digoxigenin were added tocentrifuge tubes containing 50 μl of the indicated concentration ofdigoxigenin-labeled DNA standards. The tube was centrifuged briefly topellet the cells at the bottom of the tube, nearest the PMT, and photonemission as a function of time recorded. Three different types ofdigoxigenin-labeled DNA were used to stimulate the cells, and each wassuccessful with a different degree of sensitivity. FIG. 26A. Plasmid DNAdensely labeled with digoxigenin (approximately 4000 base pairs with 200digoxigenin molecules attached) was detected with a limit of detectionof approximately 1 ng/ml (50 pg absolute). FIG. 26B. DNA molecularweight standards of various sizes (81-8576 base pairs) sparsely labeledwith digoxigenin (once per 200 base pairs) were detected at 1 μg/ml (50ng absolute). FIG. 26C. DNA molecular weight standards of various sizes(8-587 base pairs) each labeled with 2 digoxigenins (one digoxigenin oneach end of the DNA molecule) were detected at 100 ng/ml (5 ngabsolute).

FIGS. 27A-B are graphs. Centrifugation of cells may decrease sensitivityto soluble antigen. Emittor cells expressing antibody againstdigoxigenin were added to centrifuge tubes containing 50 μl of theindicated concentration of digoxigenin-labeled plasmid DNA. FIG. 27A.The tubes were centrifuged briefly to pellet the cells at the bottom ofthe tube, nearest the PMT, and photon emission as a function of timerecorded. FIG. 27B. The cells and DNA were mixed manually and placedover the PMT without centrifugation.

FIG. 28 is a graph. Two complementary Dig-labeled oligonucleotides(Oligo “3” and Oligo “NEG 3”) were allowed to hybridize underexperimental conditions. The sample was diluted 1:10 with CO2I media toa total volume of 100 μl, 20 μl of cells were added, and light emissionmeasured. Dig cells express the Dig antibody, while control cells donot.

FIG. 29 is a graph. Rapid hybridization of Digoxigenin labeled, ssDNAoligonucleotides. The indicated amount of oligonucleotide “NEG3” wasadded to 8 μl of hybridization solution (50 mM NaCl, 40 mM Tris pH 7.5).1 μl of “oligo 3” was added, followed immediately by 90 μl of CO2Imedium and 20 μl of Dig CANARY cells. The tube flicked to mix, quicklyplaced into the luminometer and light output monitored. The total timebetween addition of the second oligonucleotide and placement in theluminometer (“0” on the x axis) was approximately 15 seconds.

FIG. 30 is a graph. Single stranded DNA was generated from thepBluescript phagemid, and hybridized to all 10 Dig-labeledoligonucleotides. After hybridization the reaction was diluted to 100 μlin CO2I, 20 μl of Dig cells were added, and light emission was measured.The molar ratio indicated in the legend is that of oligonucleotide totarget ssDNA. The ideal ratio in this experiments appears to be between1:2 and 1:4.

FIG. 31 is a graph. Sequence-specific detection of single-stranded DNA.Ten digoxigenin-labeled oligonucleotide probes complementary to the (+)strand of phagemid pBluescript were hybridized to the indicated amountof single-stranded phagemid DNA. Emittor cells expressing antibody todigoxigenin were added, and light output from the cells monitored on aphotomultiplier tube. Only the (+) strand of the phagemid was detected,indicating that the identification is sequence specific. In the absenceof oligonucleotide probe, single-stranded DNA did not stimulate thecells. The limit of detection in this experiment was 50 ng.

FIGS. 32 A-B are bar charts. Effects of hybridization temperature onnucleic acid detection. Single-stranded phagemid DNA was hybridized tothe indicated concentrations of probe at several temperatures, andmaximum RLU plotted. FIG. 32A. Hybridization in PBS shows maximum signalwith hybridization at 51° C., but similar signals from sampleshybridized at 47° C. and 42° C. FIG. 32B. Hybridization at 42° C.displays an increase in the signal from experiments using lowerconcentrations of oligonucleotide probe, such that 0.16 pmoles ofoligonucleotide works nearly as well as 0.63 pmoles, and the signal from0.04 pmoles was doubled.

FIG. 33 is a schematic of the strategy for sedimentation of DNA. Captureoligonucleotides are attached to the surface of a sedimentable particle.These oligonucleotides bind to a region separate from that to which theDig-oligonucleotides bind. Target NA bind to the captureoligonucleotides, and digoxigenin labeled oligonucleotides bind to thetarget. The entire complex is sedimented by centrifugation (or magneticfield), and detected by emittor cells expressing antibody againstDigoxigenin.

FIG. 34 is a graph. Sedimentation of target DNA improves sensitivity.Streptavidin-conjugated beads were saturated with biotin labeled captureoligonucleotide, and excess oligonucleotide removed by washing.pBluescript ssDNA (+strand) was incubated with the beads for 5 min at47° C. and washed. Dig labeled detection oligonucleotides were added,hybridized for 20 min at 47° C., and excess removed by washing. Beadswere resuspended in 200 ul CO2I, and 40 ul used in each assay.

FIG. 35 is a bar chart. pBS phagemid ssDNA was incubated withbiotin-labeled oligonucleotide bound to streptavidin-coated polystyrenebeads and digoxigenin-labeled oligonucleotides for 20 minutes at 47° C.in the indicated concentrations of blocking reagent. The bead bound,digoxigenin labeled target was washed 3 times in TBS (50 mM Tris, 130 mMNaCl) at room temperature. Beads were resuspended in CO2I medium,emittor cells added, and the reaction spun and light output monitored ina luminometer.

FIGS. 36A-C are graphs. FIG. 36A U937 cells exhibit an increase in FcγRI expression when treated with IFNγ. The relative expression of FcγRIon U937 cells treated with IFNγ (200 ng/ml, open green peak) oruntreated (solid purple peak) was measured by immunofluorescence. FIG.36B U937 cells express functional aequorin protein. U937 cellstransfected with the calcium-sensitive luminescent protein aequorin emitlight when treated with ionomycin (50 M). FIG. 36C Light is detectedfollowing the crosslinking of the Fc receptors on U937 cells with stableaequorin expression. U937 cells were preincubated with 10 μg/ml humanIgG, then washed and treated with goat anti-human IgG (Fab2′).

FIGS. 37A-D are graphs. U937 cells can be engineered rapidly to respondto several different pathogens or simulants. U937 cells were treated for24 h with IFNγ (200 ng/ml) to increase expression of endogenous FcγRI,and prepared for the CANARY assay. The cells were then incubated withthe following antibodies: FIG. 37A mouse anti-B. anthracis spore, FIG.37B rabbit polyclonal anti-B. anthracis spore, FIG. 37C mouse anti-F.tularensis, or FIG. 37D mouse anti-B. subtilis hybridoma supernatant.Cells were then used in the standard CANARY assay where they detected asfew as 1000 cfu B. anthracis spores with the monoclonal antibody and10,000 cfu spores with the rabbit polyclonal, as well as 10,000 cfu F.tularensis and 1,000 cfu B. subtilis spores.

FIGS. 38A-C are graphs. The rapidly engineered U937 cells are specific,and the specificity is determined by the antibody. FIG. 38A U937 cellsincubated with mouse anti-F. tularensis antibodies did not respond to10⁵ cfu of B. anthracis spores, but did to 106 cfu of F. tularensis.FIG. 38B Cells loaded with mouse anti-B. anthracis spore antibodies didnot respond to F. tularensis but did to 10⁶ cfu of B. anthracis spores.FIG. 38C The cells did not show any response to the 10⁶ cfu of F.tularensis in the absence of anti-F. tularensis antibody [10⁶ cfu F.t.(No ab)].

FIG. 39 is an illustration of a 16-channel sensor. A sensor was designedwhich allowed the simultaneous measurement of 16 samples using a singlelight-gathering channel. The sensor consists of a rotor holding sixteen1.5-ml tubes horizontally, equally distributed about its circumference,and driven by a variable speed motor about a vertical axis. A singlefixed photon-detecting element (e.g., a PMT) is positioned in the planeof the rotor just beyond the path of the tubes during rotation. In thisway, each of the tubes is sequentially and repetitively brought intoclose proximity to the PMT, allowing its light output to be sampled oneach pass. Finally, an optical switch consisting of an optical source(an infrared LED) and a detector (a phototransistor) is used to controlthe counting of detected photons and the reorganization of the data into16 fields, each associated with a specific sample.

FIG. 40 is a graph. Data from the 16-channel sensor demonstrates an LODidentical to that obtained in a single-channel instrument, except that16 samples are measured simultaneously. A single measurement consists ofthe following steps: preparing 16 samples (and/or controls) inindividual 1.5-ml tubes, introducing an aliquot of emittor cells intoeach of the tubes, installing the tubes into the rotor situated in adark box, localizing the emittor cells to the bottom of the tubes usinga brief (5 sec) centrifugal spin at high RCF (˜2000 g), reducing therotor speed to 60 rpm for the duration of the measurement (each tubebeing sampled once every second), and generating a time-series of photoncounts for each sample for display and/or input to a computer algorithmfor evaluation.

FIG. 41 is an illustration of a portable 16-channel-sensor design.

FIG. 42 is an illustration of a CANARY Disc (CD) integrated aerosolcollection and emittor cell delivery.

FIG. 43 is an illustration of an aerosol collection module cutaway withimpaction nozzle and transparent tube.

FIG. 44 is an illustration of an emittor cell delivery module with valvedelivery system.

FIG. 45 is an overview of a 16 channel sensor and results from usingsame.

FIG. 46 is an overview of the detection of toxins.

FIG. 47 is an overview of a sensor cell that expresses aequorin and ageneralized antibody receptor.

FIG. 48 is a schematic for the detection of soluble, monomeric antigens:strategy 1. A single emittor cell is engineered to express two differentantibodies against two different epitopes on the same, monomericantigen. The presence of antigen crosslinks the antibodies, stimulatingthe emittor cell to emit light.

FIG. 49 is a graph depicting the results of a cell line expressingantibodies against both B. anthracis and Y. pestis which was challengedwith each B. anthracis and Y. pestis. This clonal cell line can detectas few as 50 cfu of either B. anthracis and Y. pestis, indicating thatboth antigen-binding sites from both antibodies are expressed andfunctional.

FIG. 50 is schematic of the strategy for detection of soluble proteins.An antigen composed of two or more epitopes is detected using twoantibodies, one bound to beads (or any support that binds to multipleantibodies) and the second antibody is expressed by the emittor cell.The antigen is incubated with the antibody-coated bead, decorating itssurface with multiple antigens. The bead is them presented to theemittor cell. Because the antigen is crosslinked by the bead, theemittor cell antibodies are crosslinked and light emission stimulated.

FIG. 51 is a graph depicting the results of antibody 6E10-10,crosslinked to Protein G magnetic beads, which was incubated withvarying amounts of BoNT/A Hc for 3 hours at 4° C. Beads were washed withCO2I medium three times. Emittor cells expressing 6B2-2 antibody wereadded, the reaction was spun for 5 seconds, and the light output wasmonitored in a luminometer.

FIG. 52 is a schematic of the detection of a chemical. A peptide isisolated that binds specifically to the chemical of interest, and anantibody generated that binds specifically to the peptide-chemicalcomplex. If the peptide-chemical only forms a single functional epitope,an additional epitope can be incorporated into the peptide. As shown,this epitope is a digoxigenin molecule, but any specific epitope wouldsuffice. In the presence of chemical, the chemical-peptide complex wouldcomprise two antibody-binding sites, and could be detected in a similarmanner as protein toxins.

FIG. 53 is a schematic depicting an alternative method for detecting achemical. Two peptides are isolated that bind in tandem to the chemicalof interest. The binding of these peptides could be detected bygenerating antibodies against each peptide-chemical complex, or bytagging the peptides with antibody binding sites as shown.

FIG. 54 is a schematic of another alternative method for detecting achemical. A peptide that binds to two chemicals of interest is preparedwhich forms a chemical-peptide dimer complex. An antibody is preparedthat binds specifically to the chemical-peptide dimer complex. Thechemical-peptide dimer complex can contain two antibody binding sitessufficient to stimulate emittor cells to increase intracellular calcium,thereby resulting in photon emission by the emittor molecule.

DETAILED DESCRIPTION OF THE INVENTION

The invention described herein provides methods for detecting solubleantigens. For example, the soluble antigen can be a soluble protein or achemical. In one embodiment, the soluble antigens comprise only one ortwo antigenic epitopes. Detection of soluble antigens using an antibodyexpressed on the surface of a cell, whereby binding of the antibody tothe antigen triggers an increase in calcium concentration which in turnstimulates an emittor molecule to emit a photon in response to theincrease in intracellular calcium depends on the ability of the antigento crosslink (or aggregate, thereby immobilizing the antibody on thecell surface) the antibodies on the cell surface, thereby stimulating anincrease in intracellular calcium. A soluble antigen can be inefficientat crosslinking antibodies expressed on the surface of a cell, andtherefore is inefficient at stimulating an increase in intracellularcalcium. Described herein are methods for detecting a soluble antigenwherein crosslinking of antibodies is achieved by the methods described,which stimulate an increase in intracellular calcium and cause emissionof a photon from an emittor molecule that responds to the increase incalcium concentration.

The soluble antigens and chemicals of interest to be detected include awide variety of agents. For example, and without limitation, the methodsof the invention described herein can be used to detect protein toxinssuch as Botulinum toxins, serotypes A, B, C, D, E, F, G, Staphylococcalenterotoxin-B (SEB) and other superantigens, ricin, pertussis toxin,Shiga toxin, conotoxins, Clostridium perfringens epsilon toxins,Shiga-like ribosome inactivating proteins, other soluble bacterialproducts, such as F1 antigen from Y. pestis, protective antigen, Lethalfactor, edema factor from B. anthracis. Other molecules of interest indetecting include bacterial quorum sensing molecules, e.g., homoserinelactones. Examples of chemical warfare agents, or their breakdownproducts after hydrolysis that can be detected using the methodsdescribed herein include, without limitation, cyanide (Hydrocyanicacid), Phosgene (Carbonic dichloride), CK (Cyanogen chloride), CL(Chlorine), CX (Carbonimidic dichloride, hydroxy), DP (Carbonochloridicacid, trichloromethyl ester), GA, Tabun (Dimethylphosphoramidocyanidicacid, ethyl ester), GB, sarin 9-Methylphosphonofluoridic acid,(1-methylethyl)ester), GD, Soman (Methylphosphonofluoridic acid,1,2,2-trimethylpropyl ester), GF (Methylphosphonofluoridic acid,cyclohexyl ester), Mustard (1,1′-Thiobis[2-chloroethane]), HN-1,Nitrogen Mustard (2-Chloro-N-(2-chloroethyl)-N-ethylethanamine), HN-2,Nitrogen mustard (2-Chloro-N-(2-chloroethyl)-N-methylethanamine),Lewsite ((2-Chloroethenyl)arsonous dichloride), PFIB(1,1,3,3,3-pentafluoro-2-trifluoromethyl-1-propene), Triphosgene(Carbonic acid, trichloromethyl ester), V-gas (Methylphosphonothioicacid, S-[2-(diethylamino)ethyl]O-2-methylpropyl ester), VX(Methylphosphonothioic acid, S-[2-[bis(1-methylethyl)amino]ethyl]O-ethylester), binary components of VX (O-Ethyl O-2diisopropylaminoethylmethylphosphonite and Sulfur), binary components of GD (Methylphosphonyldifluoride (DF) and a mixture of pinacolyl alcohol and an amine, binarycomponents of GB (Methylphosphonyl difluoride (DF) and a mixture (OPA)of isopropyl alcohol and isopropyl amine. Additionally, otherbiologically-derived chemicals can also be detected by the methods ofthe present invention, including Mycotoxins, particularly trichothecene(T2) mycotoxins, Diacetoxyscirpenol Diverse group, Saxitoxin, or otherdinoflagellage products, Microcystins (various types), Palytoxin,Satratoxin H, Aflatoxins, and Tetrodotoxin.

Additional proteins of interest to detect include, APP (AmyloidPrecursor Protein), prion proteins associated with CJD, BSE, Scrapie,Kuru, and PSA (prostate specific antigen). Furthermore, the detection ofappropriate soluble antigens or chemicals is useful in a variety ofapplications, such as clinical applications, for example, thyroidfunction, adrenal function, bone metabolism, fertility, infertility,IVF, pregnancy, growth and growth hormone deficiency, diabetes,hematology, cardiac function, cancer, allergy, autoimmune diseases,therapeutic drug monitoring, drugs of abuse, research immunoassayapplications, genetically engineered proteins, milk drug residue, liverfunction, antibiotics and antibiotic synthesis pathways. Suitablesoluble antigens for analysis in these applications are known by thoseof skill in the art (see, for example, The Immumoassay Handbook” (secondedition), David Wild, ed. Nature Publishing Group 2001. NY N.Y.).

The present invention also provides for the detection and identificationof specific nucleic acid (NA) sequences. In one embodiment, antigens areattached to the target NA using oligonucleotide probes. These probesdecorate specific NA sequences with antigen(s). This antigen-decorated(also referred to herein as antigen-conjugated) oligonucleotide iscapable of stimulating emittor cells expressing antibody against thatantigen. Free probe, if present, is monomeric, and therefore does notstimulate emittor cells. Likewise, background binding of labeledoligonucleotide to nonspecific sites on NA will not significantlystimulate the emittor cells, because the antigens resulting from theserare background binding events will be too disperse to effectivelycrosslink antibodies.

The choice of antigen depends on many factors, including theavailability and characteristics of corresponding antibodies, theabsence of crossreactive antigens in the samples to be tested, and thesolubility, stability, and cost of the antigen-oligonucleotideconjugate, as will be understood by one of skill in the art. As usedherein, an oligonucleotide can be DNA, RNA, peptide nucleic acid (PNA),locked nucleic acids, or any variety of modified nucleic acid surrogatesthat have specialized and unique characteristics as is known in the art.Additionally, the addition of cationic amino acids (in peptide orprotein form) to such probes can increase hybridization rates. Ifdesired, those cationic peptides/proteins could serve double-duty as theantigen detected by the emittor cell. Therefore, in one embodiment ofthe invention, a detection system based on emittor cells having one ormore antibodies on their surface and comprising a compound (an emittormolecule) that emits a photon upon stimulation by antigens that aremultimeric due to the presence of target NA, in particular, photonemission is stimulated by an increase in intracellular calciumconcentration.

Also provided in the invention described herein is a sensor cell thatdetects a target particle that is bound by one or more antibodies.Specifically, the sensor cells comprise an emittor molecule and an Fcreceptor that binds to an antibody which is bound to the target agent orparticle. In one embodiment, the sensor cell comprising an Fc receptoris a macrophage cell, such as the human macrophage cell line U937. Othersuitable cells or cell lines will be known to those of skill in the art.The Fc receptors are a family of membrane-expressed proteins that bindto antibodies or immune complexes. They are expressed on severalhematopoietic cells including monocytes and macrophages. Severalsubclasses of Fc receptors exist including Fc gamma Receptor I (FcγRI),a high-affinity binder of soluble antibody. FcγRI binds to the constantregion (Fc portion) of Immunoglobulin G (IgG) leaving theantigen-binding region of the antibody free. Crosslinking of theantibody-bound Fc receptor by specific antigen initiates a signalingpathway that stimulates calcium release. Therefore, crosslinking of theFc receptor on the sensor cell results in an increase in intracellularcalcium concentration and the emittor molecule thereby emits a photon inresponse to the increase in calcium concentration.

Also provided in the invention described herein is a 16-Channel Sensor.In its simplest form, an emittor cell assay consists of preparing asample in a transparent tube, introducing an aliquot of speciallyprepared emittor cells into the tube, driving the emittor cells to thebottom of the tube using a quick centrifugal spin, and measuring thelight output from the tube with a photon-counting sensor. In thelaboratory, most emittor cell assays are made sequentially, one sampleat a time; in the automated BAWS/CANARY instrument, four samples aremeasured simultaneously, each sample having its own light-gatheringchannel. The former system requires more time, while the latter requiresmore complex (and expensive) hardware.

A different approach that reduces the time to measure multiple samples(while keeping the hardware requirements minimal) is described herein. Asensor has been designed that allows the simultaneous measurement of aplurality of samples using a single light-gathering channel. The sensorconsists of a rotor holding sixteen 1.5-ml tubes horizontally, equallydistributed about its circumference, and driven by a variable speedmotor about a vertical axis (FIG. 39). A single fixed photon-detectingelement (for example, a PMT) is positioned in the plane of the rotorjust beyond the path of the tubes during rotation. In this design, eachof the tubes is sequentially and repetitively brought into closeproximity to the photon-detecting element, allowing its light output tobe sampled on each pass. Finally, an optical switch consisting of anoptical source (an infrared LED) and a detector (a phototransistor) isused to control the counting of detected photons and the reorganizationof the data into the 16 fields, each associated with a specific sample.

A further implementation of this 16-channel design is referred to as aTCAN sensor. The TCAN (Triggered-CANARY) biosensor is an automatedbiosensor which combines both aerosol collection and emittor cell liquiddelivery into an integrated radial disc format. The TCAN CANARY disc(CD) (FIG. 42) interfaces with a manifold assembly which splits an airflow into separate channels. The aerosol collection assembly (FIG. 43)uses dry impaction techniques to then localize particles from the airflow into the bottom of clear plastic tubes.

After impaction of aerosol particles, the CD interfaces with themanifold assembly to actuate valves located in the disc. The disc israpidly spun, which in turn causes the emittor cell liquid to deliver toindividual tubes using centrifugal force (FIG. 44). An optical detectoris then used to identify potential bioagents based on the photon outputof emittor cells interacting with the aerosol particles. This process ofaerosol collection and emittor cell delivery can be repeated severaltimes in one disc. This feature allows multiple emittor cell assays tobe performed after several trigger events without changing the CD.

The materials and procedures suitable for use in the invention aredescribed in further detail below.

Emittor Cells

The emittor cell (also referred to herein as a sensor cell or a CANARYcell) can be any prokaryotic or eukaryotic cell that has a suitablereceptor, signaling pathway, and signal output method, either naturally,through genetic engineering, or through chemical addition. The cell canbe an artificial or nonliving unit provided that it has a functionalreceptor, signaling pathway, and signal output method. Upon binding ofantigen receptor, such as to the antibodies, the cell mobilizes calciumions into the cytosol. An example of a cell useful in the device andmethods of the invention is a B cell (i.e., a B cell from a cold orwarm-blooded vertebrate having a bony jaw) which can be geneticallyengineered to express one or more surface-bound monoclonal antibodies.Another example of a cell useful in the device is a macrophage cell,such as the human cell line U937, which expresses an Fc receptor on thecell surface. An antigen can be bound to an antibody by addition of theantibody to the target and this antigen-antibody complex will bind tothe Fc receptor on the cell and stimulate signaling which results in anincrease in intracellular calcium.

A monoclonal antibody can be produced by, for example, immunizing ananimal with the antigen to be detected and harvesting the B cell fromthe immunized animal. DNA encoding the monoclonal antibody can then beisolated and transferred into an immortalized cell line and the cellsscreened for production of a surface monoclonal antibody specific forthe antigen to be detected. B cells are useful for both qualitative andquantitative analyses, particularly because the emission signal fromthem typically does not significantly diminish as additional targetspecimen is exposed to it and also because such emission signal islinear.

Alternatively, the cell can be a fibroblast. However, fibroblasts do notcontain the signal transduction machinery necessary to transfer a signalfrom the cytoplasmic portion of a surface antibody to calcium stores inthe cell. To overcome this problem, a chimeric surface antibody can beexpressed in the fibroblast. This chimeric antibody contains acytoplasmic amino acid sequence derived from a polypeptide (e.g., afibroblast growth factor receptor) that can transduce a signal from theinner surface of the plasma membrane of the fibroblast to intracellularcalcium stores. Thus, when an antigen binds to the extracellular portionof the chimeric antibody to cause antibody aggregation on the surface,calcium mobilization is induced. A similar strategy using chimericantibodies can be employed for any other cell type which is not a Bcell, so that the cell is suitable for use in the devices and methods ofthe invention.

Cells useful in the devices and methods herein are those designed torecognize a specific substance, including those having receptors ontheir surface that specifically bind to that substance. A preferredreceptor is an antibody or single-chain antibody, although othersuitable receptors include a mitogen receptor (such as alipopolysaccharide (LPS) receptor), a macrophage scavenger receptor, a Tcell receptor, a cell adhesion molecule, a DNA binding protein such aspart of a sequence-specific restriction enzyme or transcription factor,single-stranded-RNA- or double-stranded-RNA-binding protein, anoligonucleotide complementary to a DNA or RNA sequence to be recognized,or other ligand-binding receptor (e.g., Fas; cytokine, interleukin, orhormone receptors; neurotransmitter receptors; odorant receptors;chemoattractant receptors, etc.) that will specifically bind thesubstance to be recognized. The receptor can be attached to the cellsurface via a transmembrane domain, a membrane-bound molecule thatspecifically binds to the receptor (such as Fc receptors bind toantibodies), or a covalent or noncovalent attachment (e.g.,biotin-streptavidin, disulfide bonds, etc.) to a membrane-boundmolecule. The receptor can also be a chimeric molecule; for instance, itcan have an extracellular domain such as an antibody, single-chainantibody, lectin or other substance-specific binding domain or peptide,and an intracellular domain such as that from the insulin receptor,fibroblast growth factor, other protein that triggers a second messengercascade, etc. Instead of directly binding to the substance to berecognized, the receptor might specifically bind to another molecule orobject that in turn specifically binds to the substance to berecognized, such as a secondary antibody, labelled bead,antigen-conjugated oligonucleotide, etc.

Alternatively, only one of these binding steps may need to be specific.For instance, DNA or RNA containing specific sequences may be pulled outof solution using oligonucleotide probes conjugated to one antigen (ordirectly to a bead, or on a matrix), and a second set of nonspecificantigen-conjugated oligonucleotide probes annealed to the target DNA/RNAwould be used to stimulate cells specific for that second antigen. Also,non-specific nucleic acid binding proteins (histones, protamines,RNA-binding proteins) expressed as chimeras on the cell surface, orantibodies against those binding proteins, could also be used to detectthe presence of nucleic acids after a sequence specific selection step.

Antibodies

Whatever original cell type, the antigen-binding variable regions ofmonoclonal antibodies can obtained either as DNA sequence from a publicsource, or cloned by RT-PCR from a hybridoma cell line. RT-PCR isaccomplished using sets of primers designed to anneal, at the 5-primeend, to either the leader or framework regions of the variable region,and at the 3-prime end to the constant region.

The antibody variable regions are then cloned into expression vectorsthat already contain the constant regions for light and heavy chain. Thelight chain expression vector described in Persic et al., Gene 187:9-18,1997 is especially suitable for this purpose. VKExpress, described inPersic et al., contains the EF-1α promoter, a leader sequence, multiplecloning sites, and the human Ig kappa constant region andpolyadenylation signal. The heavy chain expression vector is derivedfrom Invitrogen's pDisplay. This vector contains a CMV promoter, aleader sequence, an HA tag, multiple cloning site, and myc tag, followedby the PDGFR transmembrane domain and bovine growth hormonepolyadenylation signal.

pDisplay can be modified for heavy chain expression as follows. ThePDGFR transmembrane domain of pDisplay is replaced with the murine IgMconstant region without the exon that allows for secretion. This ensuresthat the protein will remain membrane-bound. The neomycin-resistancegene can be replaced by any of a number of antibiotic-resistance genesincluding, but not limited to, hygromycin, bleomycin, puromycin,kanamycin, and blasticidin genes. The heavy chain (or alternativelylight chain) variable region can be inserted in a two-step process,using overlap-extension PCR, to remove the HA and myc tags present oneither side of the multiple cloning site of pDisplay. A vector can alsobe developed to allow insertion of an overlap extension productcontaining the variable region fused to approximately 300 base pairs ofthe IgM constant region, so that cloning can be done in a single step.

The examples below were implemented using the antibody vectorconstruction procedure described immediately above.

An antibody which specifically binds to the antigen to be detected is amolecule which binds to the antigen or an epitope of the antigen, butdoes not substantially bind other antigens or epitopes in the sample.Such antibodies can be chimeric (i.e., contain non-antibody amino acidsequences) or single chain (i.e., the complementarity determining regionof the antibody is formed by one continuous polypeptide sequence).

Alternatively, surface antibody-producing cells can be obtained from theanimal and used to prepare a monoclonal population of cells producingsurface antibodies by standard techniques, such as the hybridomatechnique originally described by Kohler et al., Nature 256:495-497(1975); Kozbor et al., Immunol Today 4:72 (1983); or Cole et al.,Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc., pp. 77-96(1985). The technology for producing cells expressing monoclonalantibodies is well known (see, e.g., Current Protocols in Immunology(1994) Coligan et al. (eds.) John Wiley & Sons, Inc., New York, N.Y.),with modifications necessary to select for surface antibodies ratherthan secreted antibodies.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating acell producing a surface monoclonal antibody (see, e.g., CurrentProtocols in Immunology, supra; Galfre et al., Nature 266:55052, 1977;Kenneth, In Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y., 1980; and Lerner,Yale J Biol Med 54:387-402 (1981). Moreover, the ordinarily skilledworker will appreciate that there are many variations of such methodswhich also would be useful.

Polyclonal cells expressing antibodies can be prepared by immunizing asuitable animal with the antigen to be detected. The cells producingantibody molecules directed against the antigen can be isolated from theanimal (e.g., from the blood) and further purified by well-knowntechniques, such as panning against an antigen-coated petri dish. As analternative to preparing monoclonal cells, a nucleic acid encoding amonoclonal antibody can be identified and isolated by screening arecombinant combinatorial immunoglobulin library (e.g., an antibodyphage display library) with the antigen to thereby isolateimmunoglobulin library members that bind the antigen. Kits forgenerating and screening phage display libraries are commerciallyavailable (e.g., the Pharmacia Recombinant Phage Antibody System,Catalog No. 27-9400-01; and the Stratagene SurfZAP® Phage Display Kit,Catalog No. 240612). Additionally, examples of methods and reagentsparticularly amenable for use in generating and screening antibodydisplay library can be found in, for example, U.S. Pat. No. 5,223,409;PCT Publication No. WO 92/18619; PCT Publication No. WO 91/17271; PCTPublication WO 92/20791; PCT Publication No. WO 92/15679; PCTPublication WO 93/01288; PCT Publication No. WO 92/01047; PCTPublication No. WO 92/09690; PCT Publication No. WO 90/02809; Fuchs etal., Bio/Technology 9:1370-1372 (1991); Hay et al., Human AntibodHybridomas 3:81-85 (1992); Huse et al., Science 246:1275-1281 (1989);Griffiths et al., EMBO J. 12:725-734 (1993).

After the desired member of the library is identified, the specificsequence can be cloned into any suitable nucleic acid expressor (e.g., avector) and transfected into a cell such as a fibroblast. The expressorcan also encode amino acids operably linked to the antibody sequence asappropriate for the cell which is to express the antibody. As discussedabove, the cytoplasmic transmembrane sequence of a fibroblast growthfactor receptor can be linked to a single-chain antibody specific forthe antigen to be detected, so that the cell immobilizes calcium whencontacted with the antigen. Although separate recombinant heavy chainsand light chains can be expressed in the fibroblasts to form thechimeric antibody, single chain antibodies also are suitable (see, e.g.,Bird et al., Trends Biotechnol 9:132-137, 1991; and Huston et al., IntRev Immunol 10:195-217, 1993).

Photon Emitter Molecules

Binding of the desired substance to the cell-surface receptor shouldtrigger a signaling pathway inside the cell. A preferred signalingpathway is the second-messenger cascade found in B cells, T cells, mastcells, macrophages, and other immune cells, wherein crosslinking of thecell surface receptors activates a tyrosine kinase, which thenphosphorylates phospholipase C, which then cleaves phosphatidylinositol4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) anddiacylglycerol; IP3 then opens calcium channels to release calcium fromintracellular stores such as the endoplasmic reticulum or to let inextracellular calcium, thereby elevating the calcium concentration inthe cell's cytosol. Depending on the receptor type, cell type, anddesired signaling method, alternative second-messenger cascades could beemployed, such as a G-protein-adenylyl cyclic-cAMP-protein kinase Acascade.

A method should be provided for monitoring the internal signaling of thecell in response to substances to be identified. If the internalsignaling involves an increase in cytoplasmic calcium, a preferreddetection method is a calcium-sensitive luminescent or fluorescentmolecule, such as aequorin, obelin, thalassicolin, mitrocomin(halistaurin), clytin (phialidin), mnemopsin, berovin, Indo-1, Fura-2,Quin-2, Fluo-3, Rhod-2, calcium green, BAPTA, cameleons (A. Miyawaki etal., (1999) Proc. Natl. Acad. Sci. 96, 213540), or similar molecules. Itis anticipated that the relative intensities of light and the sensorcell storage characteristics enabled by using calcium-sensitivemolecules may vary depending on the efficiency of light production forthe specific emitter molecule and the half-life of the activated emittermolecule—in some cases providing significant benefits (e.g., improvedsensitivity, quantitative or qualitative detection). Additionalperformance enhancements may arise from the use of structural analogs ofthe natural cofactors of photoprotein emitter molecules. Variouscalcium-sensitive fluorescent dyes which can be taken up by live cellsare available from commercial sources, including Molecular Probes, Inc.,Eugene, Oreg. Proteins such as aequorin, obelin, thalassicolin,mitrocomin (halistaurin), clytin (phialidin), mnemopsin, berovin orcameleons could be added genetically, injected into the cells, ordelivered by a protein uptake tag from HIV TAT (approximately aminoacids 47-57; A. Ho et al. (2001) Cancer Research 61, 474-477) or byother means. If desired, such reporter molecules can include targetingsignals to target them to the cytoplasmic face of the endoplasmicreticulum or the plasma membrane, the interior of the mitochondria, orother locations where the change in local calcium concentration might beparticularly large. Optical methods of detecting activity from otherpoints in the signaling pathway could also be used, such as fluorescenceresonance energy transfer (FRET) of fluorescent groups attached tocomponents of the signaling pathway (S. R. Adams et al. (1991) Nature349, 694-697). Where the internal signaling involves an increase inreactive oxygen species (e.g. superoxide anion radicals, hydroxylradicals, compound I or II of horseradish peroxidaase, etc.), apreferred detection method is a reactive-oxygen-sensitive luminescent orfluorescent molecule, such as the photoprotein pholasin (a 34-kDaglycoprotein from the bioluminescent mollusc, Pholas dactylus) orsimilar molecules. Alternatively, a reporter gene for any luciferasecould be linked to a promoter induced by the signaling pathway. In somecells such as T cells and mast cells, the signaling pathway triggersexocytosis of granules containing proteases such as granzymes,tryptases, or chyrnases. Exocytosis of these proteases could be detectedby calorimetric or fluorometric methods (e.g., p-nitroanaline or7-amino-4-trifluoromethyl coumarin (AFC) linked to peptides cleaved bythe proteases [S. E. Lavens et al. (1993) J. Immunol. Methods 166, 93;D. Masson et al. (1986) FEBS Letters 208, 84; R&D Systems]). Also,microelectrodes or other methods to detect the electrical activityassociated with the calcium flux or other signaling ion fluxes aresuitable to monitor signaling response in the cell.

A suitable emitter molecule is any molecule that will emit a photon inresponse to elevated cytosolic calcium concentrations, includingbioluminescent and fluorescent molecules. One emitter molecule, thebioluminescent aequorin protein, is described in Button et al., CellCalcium 14:663-671 (1993); Shimomura et al., Cell Calcium 14:373-378(1993); and Shimomura, Nature 227:1356-1357 (1970). Aequorin generatesphotons by oxidizing coelenterazine, a small chemical molecule.Coelenterazine diffuses through cellular membranes, so coelenterazine oran analog thereof can be added to the culture medium surrounding thecells. Alternatively, genes encoding enzymes that make coelenterazinecan be introduced into the cells. In another embodiment, bioluminescentgreen fluorescent protein (GFP) (see Chalfie, Photochem Photobiol62:651-656 [1995]) or yellow fluorescent protein (YFP) can be used. Inthis embodiment, the cell cytosol contains both GFP and aequorin. Inresponse to elevated calcium in the cytosol, aequorin donates energy toGFP in an emissionless energy transfer process. GFP then emits thephoton. Alternatively, the emitter molecule can be a calcium-sensitivefluorescent molecule (e.g., indo-1) which is illuminated by a wavelengthof light suitable to induce fluorescence.

Aequorin, or any other emitter molecule, can be introduced into the cellby methods well known in the art. If the emitter molecule is a protein(as is the case with aequorin), the cell can contain an expressionvector encoding the protein (i.e., a nucleic acid or virus which willproduce the emitter molecule when introduced into a cell). An expressionvector can exist extrachromosomally or be integrated into the cellgenome.

Conjugated Antigens/Tags

One or more antigens or tags can be added (also referred to herein asconjugated) to molecules to provide a known antigenic epitope. Forexample, one or more antigens can be conjugated to an oligonucleotide toproduce an antigen-conjugated oligonucleotide with a known antigenicepitope. An antigen-conjugated molecule can comprise one antigen ormultiple antigens that are either the same of different. For example andwithout limitation, an antigen or tag to be conjugated to a molecule fordetection includes small antigens such as digoxigenin, digoxin,phosphocholine, fluoroscein or other fluorphores, and biotin, andpeptides such as HIS, VSV-G, FLAG, and C(AAKK) multimer (as described inCorey, J. Am. Chem. Soc., (1995) 117: 9373-4).

Oligonucleotides

In addition to conventional DNA and RNA probes, a variety of modifiednucleic acids have been shown to hybridize in a sequence-specific mannerto target nucleic acid sequences. These include peptide nucleic acids(PNA) (Nielsen et al., (1991) Science 254: 1497-1500), Bis-PNAs(Griffith et al., (1995) J. Am. Chem. Soc 117: 831-832), Tail-clamp PNA(Bentin (2003) Biochemistry 42: 13987-13995), PD loops (Bukanov et al.,(1998) PNAS 95: 5516-5520), PNAs incorporating pseudocomplementary bases(Lohse et al., (1999) PNAS 96 (21) 11804-11808), or locked nucleic acids(Braasch and Corey (2001) Chem. Biol. 8: 1-7). A variety of thesemodified nucleic acids have been shown to have differ in hybridizationcharacteristics, stability, affinity, and specificity, and could be usedin place of conventional DNA oligonucleotides (reviewed by Beck andNielsen, pp. 91-114, in Artificial DNA: Methods and Applications. CRCPress, Y. E. Khudyakov and H. A. Fields eds.). Attachment of cationicproteins, peptides, or DNA binding proteins has been shown to improvehybridization kinetics (Corey (1995) J. Am. Chem. Soc 117: 9373-9374;Zhang et al., (2000) Nuc. Ac. Res. 27 (17) 3332-3338).

The binding of oligonucleotides has been shown to improve with theaddition of helper oligonucleotides (O'Meara et al., (1998) Anal.Biochem. 225: 195-203; Barken et al, Biotechniques (2004) 36: 124-132).Specificity can be improved by addition of unlabeled hairpin competitorprobes (Huang et al., (2002) Nucleic Ac. Res. 30: (12) e55).

Removal of unbound oligonucleotides after hybridization to target is notnecessary for nucleic acid sequence detection, but may be desirable. Theunbound labeled oligonucleotide could be removed using a variety ofconventional chromatography techniques, including size exclusion,hydrophobic interaction, or ion exchange, depending on the chemistry ofthe particular probe used.

Other Nucleic Acid-Binding Molecules

Oligonucleotides are not the only molecules that are able to identifyspecific nucleic acid sequences. Proteins are also capable of suchdiscrimination, and can be expressed on the surface of the emittor cell,recombinantly attached to a cytoplasmic domain that would, upon binding,initiate a calcium response. This would include nucleic acid bindingproteins attached to the Fc portion of antibodies, for example.Expression of nucleic acid binding proteins on the surface of theemittor cell would eliminate having to denature double-stranded nucleicacid prior oligonucleotide hybridization, and additionally, the systemproduces all the necessary components: no exogenously synthesizedoligonucleotides would be required. Possible sequence specific DNAbinding proteins include: (1) DNA restriction enzymes (preferably withthe DNA-cutting catalytic site removed or inactivated, e.g. L. F. Dorner& I. Schildkraut (1994) Nucl. Acids Res. 22, 1068-1074); (2)Transcription factors or other specific DNA- or RNA-binding proteins,especially those that recognize unique DNA or RNA sequences in pathogensor organisms of interest (e.g., HIV TAT transcription factor: C. Brigatiet al. (2003) FEMS Microbiology Letters 220, 57-65; poxvirustranscription factors: S. S. Broyles (2003) Journal of General Virology84, 2293-2303). Emittor cells with such receptors could be designed tocrosslink on target DNA/RNA with either a specific repeated sequence oralternatively two or more unique sequences.

Capture Oligonucleotides

Although not necessary for detection, capture of the target nucleic acidsequence on sedimentable or solid support can improve assay sensitivity.Single-stranded DNA target can be captured using, for example,biotin-labeled capture oligonucleotides bound to streptavidin-coatedpolystyrene or paramagnetic beads. The captured material can beseparated from unbound material by centrifugation or exposure to amagnetic field, as appropriate. The use of an intermediate bindingreaction (avidin-biotin) in attaching the oligonucleotide to the beadmay not be necessary as any interaction that would attach theoligonucleotide to a solid support can be used, including directconjugation. In addition, any solid support to which the captureoligonucleotide can be attached would suffice. This can be in the formof a two-dimensional array, in which specific capture oligonucleotidesare placed in specific positions on the array. Alternatively, targetnucleic acid sequences can be captured in a non-specific manner (e.g.ion exchange resin, precipitation, histone or protamine binding). Targetcapture will also concentrate the target nucleic acid sequence and/orremove assay interferents.

Polyvalence

Emittor cell stimulation is dependent on the antigen appearingmultivalent to the emittor cell. In general, this can be accomplished inat least two ways. First, multiple copies of antigen can be attached toa target molecule, for example, hybridizing multiple antigen-conjugatedoligonucleotides to the target nucleic acid sequence. Second, severalcopies of the target nucleic acid sequence, each with a single antigenattached, can be bound to each other or bound in close proximity to eachother (e.g., attached to a bead). In this example, the individual targetnucleic acid sequence would not be polyvalent, but the bead withmultiple copies of the target nucleic acid sequence attached wouldpresent a polyvalent antigen.

Reaction Chambers

The reaction chambers suitable for use in the invention can be anysubstrate or vessel to which emitter cells and candidate particles canbe mixed and contacted to each other. In one embodiment, the reactionvessel is a centrifuge tube (e.g., a microcentrifuge or Eppendorf tube).As described herein, centrifugation is a particularly well-suited meansto pellet candidate particles or emitter cells first, before the otheris driven into the first pellet. To further increase the pelleting ofboth particles and cells, the side walls of the tube can be coated witha non-sticky carrier protein such as bovine serum albumin to prevent thesticking of emitter cells to the side walls, and the bottom of the tubecan be coated with poly-L-lysine to help ensure that the targetparticles stay adhered to the bottom of the tube. Other proteins ormolecules that either prevent or promote cell adhesion are known in theart of cell biology and are suitable for use in the invention.

Centrifuge tubes with customized sample well geometries can provide anadditional embodiment that uses centrifugation to increase emittor cellinteractions with difficult-to-sediment particles and reduces the needto customize spin sequence. In this embodiment the particle-containingsample to be analyzed is placed in a tube where the maximum width of thesample chamber is approximately equal to the diameter of an emittercell. Layering a concentrated emitter cell suspension over the samplefollowed by centrifuging drives a large number of closely packed emittercells through the smaller particles while the constrained geometryincreases the probability of emitter cell antibody interaction withparticles. Binding of the cell-associated antibody to the particlecaptures the poorly sedimenting particle and will rapidly draw it to thebottom of the tube with the emitter cell where the resulting light canbe observed by a photo multiplier device.

In another embodiment, the reaction chambers are wells in atwo-dimensional array, e.g., a microtiter plate, or spots or wells alonga tape, as shown in the figures. These arrangements allow multiplexdetection of either multiple samples and/or multiple target particles.For automated delivery of candidate particles and/or emitter cells,either the reaction chambers or the specimen collector and emitter cellreservoir is addressable in at least two dimensions. The wells of arrayscan also be treated with sticky and non-sticky coatings as describedabove for centrifuge tubes to facilitate contact between emitter cellsand candidate particles.

Specimen Collectors

Different devices can be used to collect samples from, e.g., air. Ingeneral, an air sampling device has a collection chamber containingliquid through or beside which air or gas is passed through, orcontaining a porous filter that traps particulates (e.g., targetparticles) as air or gas passes through the filter. For collectionchambers containing liquid, the collection liquid can be centrifuged orotherwise treated to separate particles from the liquid. The separatedparticles are then deposited in a reaction chamber. For collectionchambers containing a filter (e.g., nitrocellulose), the filter orportions of the filter can act as the reaction chamber. Alternatively,particles can be washed from the filter, or the filter can be dissolvedor otherwise removed from the particles. A filter collection chamber canalso be adapted to collect particles from a liquid (e.g., water supplysample or cerebral spinal fluid) flowing through the filter. Inaddition, as discussed above, a liquid sample can be centrifuged toremove any particulate material present in the liquid. A variety ofsamplers are known and available for use with the present invention. SeeSKC, Inc., which sells the SKC BioSampler®. and other sampling devices.

Other air samplers can be used. For example, an alternative device isthe Air-O-Cell sampling cassette (SKC, Inc.). In this device, theairborne particles are accelerated and made to collide with a tackyslide which is directly suitable for various staining procedures andmicroscopic examination.

Aerosol particulates may be collected using inertial separation in adevice known as an impactor. An airflow containing particles to becollected is drawn from the environment of interest into the impactorwhere it is directed towards a surface for impaction. With appropriategeometrical parameters and flow rates in the impactor, particles withsufficient inertia will not follow the flow streamlines, but will impactonto the surface. A significant proportion of the particles impactingthe surface adhere through electrostatic and/or van der Waalsinteractions and are thereby collected and concentrated. In this way,aerosol particles containing proteins (including toxins), viruses,bacteria (vegetative and spore forms), parasites, pollen and otherdetectable substances can be collected for detection using a variety ofavailable assay technologies including the devices and methods herein.

Dry sample collection for bioassays using an air impactor providesgeneral advantages over traditional air-to-liquid sample collection byreducing or eliminating fluid consumables and transfer mechanisms whichreduces assay cost and simplifies automation. Of particular benefit tothe devices and methods herein, collection using dry impaction ensuresthat all of the collected sample is located on the surface prior to theaddition of sensor cells of the devices and methods herein, regardlessof the size of the individual analyte particles. This achieveslocalization of all analytes regardless of their sedimentationcoefficient in fluid, thereby maximizing the sensitivity of the devicesand methods herein and accelerating many implementations of the assay byeliminating a time-consuming step.

Any surface that retains a proportion of particles that impact onto itand that is compatible with subsequent bioassays is suitable as acollection surface. Suitable materials include biocompatible metals,plastics, glasses, crystals, aerogels, hydrogels, papers, etc.Particularly useful configurations of these materials includemicrocentrifuge tubes, multi-well plates used in high-throughputscreening, continuous tapes, filters, conjugate release pads of lateralflow immunoassays, etc. The collection efficiency can be increased bymodifications to the collection surface including: the addition ofcoatings promoting adhesion of biological particles (these coatings canbe chemical or biochemical in nature, e.g. polylysine), increasedsurface roughness to increase the surface area available for collection,and customized surface geometries that promote deposition of particlesin defined regions on the surface. Furthermore, additional improvementsin collection efficiency can be achieved by manipulating theelectrostatic charges on the collection surface and the incomingparticles such that additional attractive forces are generated.

Additional improvements can be made to the dry impaction collector byusing an air-to-air concentrator upstream of the collector to increasethe number of particles in each unit of air sample impacted onto thecollection surface. This can significantly reduce the amount of timeneeded to collect a sufficient number of aerosol particles to providereliable results for the detector.

In one example of this collection concept, the impactor described inFIG. 23 has been configured to collect aerosol samples on the bottom ofa commercially available plastic tube. A nozzle projects down into thetube and the exit is positioned at the radius of curvature of the tube'sinner surface. This positioning increases the likelihood of particleimpaction upon the tube bottom where the device sensor cells are mostlikely to contact them. Once collection is completed, a single dropletcontaining device sensor cells is added directly to the tube containingcollected aerosol particles, spun for 5 seconds to accelerate celldelivery to the tube surface, and emitted light is measured using aphoton detector (e.g., PMT, CCD, photodiode, etc.). Using thisapparatus, dry bacterial spores can be collected from an aerosol andidentified directly with optoelectronic device in less than one minute.This method can be implemented with a plurality of tubes used to collectsamples and an automated system to conduct subsequent assays. An exampleof how a system capable of conducting at least 10 independent assays isshown in FIGS. 4, 6, 9, 12, and 15. By implementing an approach whereassays are made capable of looking for multiple analytes in a singletube (multiplexed) the number of detectable substances for a singleassay cycle can be made greater than the number of available tubes. Thiscan be done by creating individual optoelectronic detection device celllines expressing a plurality of receptors with affinity for differentanalytes or by combining multiple cell lines with differentspecificities in a single tube.

FIG. 4 is a schematic of an integrated biological aerosol warning sensor(BAWS)/optoelectronic sensor system. The BAWS trigger module is used topreliminarily detect the presence of particles, e.g., those of apre-determined size range. If particles meeting specifications aredetected, BAWS triggers an air-to-air concentrator that allows particlesof a particular size range to be collected and deposited in a well(e.g., reaction chamber, tube) via a dry-impactor module. Thedry-impactor module allows for dry sample collection and is incommunication with a syringe module for cell (e.g., emitting cells)delivery into a reaction chamber (e.g., tube). A transport module isused to transfer the reaction chamber assembly (having one or morechambers or tubes) to a centrifuge module for sedimentation or mixing ofthe particle sample and cells. The centrifuge module can be, but neednot necessarily be, in communication with an optics/PMT module fordetection of photon emission. A controller module is useful for controlof operation of the system.

FIG. 6 shows an example of a dry-impactor module concept. In thisexample a single (e.g., prototype system) as well as a multi-channeldevice is illustrated, including individual sample tubes (e.g., PCRtubes) and tube carriers, in communication with air-to air concentratorsfrom which the particle test sample is collected.

FIG. 9 shows an example of a cell-delivery that can be automated. Thesensor cells (e.g., emitting cells) are introduced to the system bymeans of a syringe and syringe pump arrangement, which can includepipettors or other delivery equipment. This type of assembly allows formultiple and simultaneous introduction of sensor cells to the particlesamples (e.g., samples in reaction chambers (e.g., tubes).

FIG. 12 shows an example of a centrifuge module concept used to spin theparticle samples or cell samples. Carriers having the sample tubes areintroduced via a loading mechanism into a rotor assembly that issuitable for receiving the carriers. The rotor spins the samples. Therotor assembly is in communication with optics modules for signalcollection (e.g., photon emission), and an indexed motor can be used toallow for alignment of the samples chambers with the detector (e.g.,optics modules).

FIG. 15 shows an example of an optics module. Depending on the preciseconfiguration, the module allows for a plurality of simultaneous testingof samples (e.g., in the reaction chambers, tubes). The carrier andtubes therein are introduced to the unit such that they are incommunication with lens assemblies (e.g., integrated reflectors, lenses)if necessary, and ultimately a photodetector (e.g., a PMT). The PMTproduces signals that are then sent to a processor for processing anddisplay.

FIG. 21 illustrates an integrated dry-impactor/optoelectronic sensor. Inthis sensor the modules described above are assembled in a lineararrangement with a cassette holding 30 carriers deliverable to abelt-driven carrier transport module. This transport module moves theassay tubes sequentially from the collector to the cell delivery moduleto the centrifuge module, and finally to the confirmatory sample storagemodule following completion of photon detection. The overall size ofthis integrated sensor is approximately 54 inches wide by 33 inches highby 22 inches deep.

Real-world samples may contain substances that either inhibit the assay(false negative) or cause a response in the absence of specific antigen(false positive). In many instances, these samples can be treated priorto the assay to remove these substances. For example, soluble substancessuch as detergents or serum factors can be removed by pre-centrifugationstep, where the agent is concentrated in the bottom of the tube and theliquid is replaced with assay medium (Portal Shield samples). Insoluble,large particulate substances can be removed from the sample byfiltration, using commercial filters of a pore size (3-5 μm) that allowsthe passage of the agent, but retains the contaminant (diesel or sootsamples). Samples can be processed rapidly through syringe filters,adding only a few minutes to the total assay time.

Specimen Localization

As part of the specimen collector or reaction chamber, differentmechanisms (other than centrifugation) can be implemented to facilitatecontact between emitter cells and candidate particles. For example, theuse of electrophoresis, isoelectric focusing, dielectrophoresis,magnetically tagged particles, and the like in bioelectronic devices canbe integrated into a system of the invention. See, e.g., U.S. Pat. No.6,017,696 and other patents assigned to Nanogen, Inc.; Goater et al.,Parasitology 117:S177-189, 1998; and U.S. Pat. Nos. 5,512,439 and4,910,148 and other patents assigned to Dynal AS.

Mixing a aqueous sample containing target particles (particles here canbe anything recognized by the emitter cells-proteins/toxins, viruses,bacteria, parasites, nucleic acids, etc.) with an aliquot of mediacontaining emitter cells results in particle-cell contact leading totransient increase in the rate of photon emission. The time between thestart of the mixing process and the maximum emission rate depends on thecharacteristic response of the particular cells to stimulation as wellas the time over which the mixing occurs (the mixing time) and thetypical time for the particles and cells to come into contact aftermixing (the diffusion time).

Because a background rate of detected photons will exist even in theabsence of target particles (background cell emission and thermal noisein the photon detector and its electronics, for example), photonsemitted from single target-cell interactions can be difficult todistinguish from this background. To be useful as a signal, there mustbe a significant increase in the rate of photons detected over that ofthe background. For a given sample, this rate is maximized when themixing time and diffusion time are minimized. Other possible signalsthat target particle are present in a sample include: an increase in thetotal number of photons detected in a period of time above that of thebackground alone, a change in the statistics of detected photons, or achange in the spectral qualities of the detected photons.

The diffusion time can be minimized by reducing the average distancebetween particle and cell after mixing. This can be accomplished bylocalizing the particles and/or cells to within a small volume, often alayer, within the larger mixed volume. However, the time to localize theparticles and/or cells may be longer than the characteristic responsetime of the cells. Mixing between particles and cells over thisprolonged localization could produce a lower rate of photon emission,and therefore a lower signal, by increasing the average time betweenemissions. To avoid this, one or both should be localized separately,while minimizing contact between them. This localization can also leadto a reduced mixing time.

Generally, the means to move particles or cells include the following:sedimentation (by gravity or centrifuge); fluid flow (forced orconvective); electric forces (electrophoresis and dielectrophoresis);magnetic forces (using magnetic beads); and acoustics/ultrasonics(standing or traveling waves).

Localization requires a means of moving particles and/or cells combinedwith a barrier where particles and/or cells can collect, such as thesolid surface of a channel or container, the surface of a filter, or thepotential energy barrier surrounding an electric-field minimum. Examplesinclude: sedimentation (localizing cells on the lower surface of achamber); air impaction (impacted particles stick to or settle onto acollection surface); filtering (particles or cells collect on to thesurface or into the body of a filter); affinity capture particles orcells can be localized through specific or non-specific bindinginteractions); magnetic capture (magnetic beads held against a solidsurface, a filter surface, or in the body of a filter by localizedmagnetic forces; beads may or may not have surface chemistry to promoteattachment of particles or cells); electrophoresis (charged particlesonly; collection on to an electrode surface); and dielectrophoresis(positive: collection of particles or cells on to an electrode surface;negative: collection into a region of minimum field).

Localization and mixing of particles and cells can be achieved bycombining the above methods, as well as others. In the table below,examples of various localization/detector combinations are provided.Certain of the representative examples illustrate methods to localizeparticles or cells 2-dimensionally, allowing improvement in sensitivityor discrimination between different particles if an array of photondetectors (including a CCD) is used as opposed to a single photondetector (such as a PMT).

Method of Method of Mixing: particles or Example localizing cellslocalizing particles cells/means Detector centrifuge centrifugecentrifuge (long) cells/sediment (cent.) single (short) flow cellsediment and shallow channel particles/sediment single attach to surfaceabove cells (grav.) flow cell (multiple sediment and shallow channelparticles/sediment imaging cell lines) attach to surface above cells(grav.) flow cell/magnetic sediment and localized magnetic particles (onimaging bead attach to surface bead capture beads)/sediment (grav.) flowcell/electric sediment and shallow channel particles/electrophoresissingle field attach to surface above cells tape/wick flow (into wick)air impact (tape) cells/sediment (grav.) single air impact centrifugeair impact (tape) cells/sediment (cent.) single (short) uniprep/magneticsediment to magnetic beads on particles (on single bead surface filtersurface beads)/sediment (grav.) flow past cells cells on filter flowpast cells single surface counter flow cells held on retained on filterparticles/flow past cells single filter surface by surface counter tocent. Force centrifugation centrifuge tube centrifuge onto retained inflow by cells/sediment (cent.) single dielectrophoretic filter surfacedielectrophorectic trap force traveling-wave sediment and traveling-waveparticles/sediment single dielectrophoresis attach to dielectrophoresis(grav.) traveling-wave dielectrophoresis dissolvable- separatecentrifuge (long) cells or single membrane tube compartment ontodissolvable particles/traveling-wave membrane dielectrophoresisacoustic/ultrasonic dissolve membrane and sediment (cent.)

LOCALIZATION EXAMPLES

In each of the following examples, it is assumed, unless statedotherwise. The sample is an aliquot of aqueous solution compatible withshort-term cell life and function, possibly containing target particles(though the descriptions below will assume the presence of particles).An aqueous sample can be obtained from environmental, clinical,air-to-liquid, washed-swab, or other samples. An air sample can beobtained from a driven air stream (air sampler or surface pickup),electrostatic capture, or settled airborne particles. References tocells should be understood to mean emitter cells in an aqueous mediathat is compatible with their life and function. A particle and cellbrought into contact is assumed to result in emission of one or morephotons. A single or array photon detector exists external to thechamber in which the sample and cells are mixed, and there may beadditional optical elements to enhance capture and detection of emittedphotons (such as mirrors, lenses, lightpipes, etc.) either external orinternal to the chamber. The chambers are either assumed to betransparent in part or in whole or to have another means to allowemitted photons to reach the detector.

Centrifuge

A sample can be centrifuged in a chamber for a time sufficient tosediment the particles. Cells can be introduced to the chamber withoutdisturbing the particles and briefly centrifuged to sediment them ontothe particles. Photon detection can occur during or, more typically,after the spin.

Affinity Capture (Surface Capture)

A sample can be introduced into a microcentrifuge tube, multi-wellplate, filter unit, or other suitable device where some portion of thesurface in contact with the sample has been modified to be able to bindand retain particles that may be present in the sample through specificor non-specific binding interactions. Non-specific binding may befacilitated via electrostatic/ion-exchange interactions, hydrophobicinteractions, hydrophilic interactions, etc. Specific binding may befacilitated by immobilizing components to the surface that bind tosubstrates on the particles (e.g. antibodies, receptors, glycoproteins,proteins, peptides, carbohydrates, oligonucleotides, etc.), or byimmobilizing components that are bound by receptors on the surface ofparticles (small molecules, peptides, proteins, carbohydrates, etc.).

Affinity Capture (Onto Mobile Substrate)

Similar to affinity capture on a surface, but particles are bound tomobile substrates (polymer beads, cells, charged molecules, magneticbeads, bacteria, etc.) that provide additional means of moving and/orlocalizing the particles or cells by various methods including thosedescribed herein.

Flow Cell

Emitter cells can be introduced to a shallow flow cell and allowed toattach to the bottom surface; non-adherent cells can be removed byadditional flow. A sample is introduced, displacing much of the cellmedia, and particles can sediment out onto the attached cells. Photonsare emitted as particles contact cells.

Flow Cell (Multiple Cell Lines)

Similar to the Flow Cell, with distinct regions of emitter cellsensitive to different target particles. Photon detection by imagingdetector to allow identification of which cells are stimulated, and,therefore, which target particles are present in the sample.

Flow Cell (Magnetic Bead)

This is similar to the Flow Cell. Appropriate magnetic beads are mixedwith the sample, allowing target particles to attach to the beads. Thesedecorated beads can be introduced to the flow cell where a stronglocalized magnetic field (due to a permanent magnet or electromagnet)captures them on the surface above the attached cells. Mixing can beinitiated by either removing the magnetic force and allow the beads tosediment onto the cells, or moving the magnetic force to attract thebeads to the surface to which the cells are attached.

Flow Cell (Electric Field)

Similar to Flow Cell, with the surface to which the cells attach and theone parallel to it being separate electrodes (at least one of whichmight be transparent). A sample can be introduced, displacing much ofthe cell media. An appropriate DC voltage is applied between theelectrodes and the particles are moved to the attached cells byelectrophoresis.

Tape/Wick

An air sample, possibly containing target particles, can be impacted ona transparent surface, which can be rigid or flexible (e.g., a tape),porous or nonporous. An absorbing material, or wick, can be attached,surrounding the impact area or, in the case of a porous surface, on theopposite side of that surface. Cells can be placed on the impact area,and, due to the wick, excess media will be absorbed, reducing the volumeand depth of the media bearing the cells and bringing them closer to theparticles. Cells sediment out onto the impacted particles or are,additionally, drawn toward them by flow if the surface is porous withthe wick material behind.

Air Impact

An air sample, possibly containing target particles, can be impactedinto a (fixed and initially empty) chamber which is suitable forcentrifugation. Cells can be introduced to the chamber withoutdisturbing the particles and briefly centrifuged to sediment them ontothe particles. Photon detection can occur without, during, or, moretypically, after the spin.

Filter Device/Magnetic Bead

A modified syringeless filter device, consisting of a chamber and aplunger with a suitable filter (Whatmanm, Mini-Uniprep™, or similar),can be loaded with cells which are allowed to attach to the bottomsurface of the chamber; unattached cells can be washed away. A samplecan be introduced to the chamber along with magnetic beads with asuitable surface affinity. A modified plunger with a suitable magnetinserted inside and fixed near the back-side of the filter can beinserted into the chamber until the entrapped air escapes through thefilter. This assembly can be inverted and (possible after a time toallow the beads to sediment onto the filter's surface) the chamberpushed down onto the plunger. Magnetic beads and particles canaccumulate on the filter surface by filtration, sedimentation, andmagnetic attraction. Particles can attach to the magnetic beads or becaught among them. Upon re-inverting the assembly, the particles, areheld off the cells by the magnetic beads which, in turn, are held by themagnet inside the plunger. Removing that magnet releases the beads, andthe particles, which sediment across the short distance onto the cells.

Flow Past Cells

One or more layers of cells can be allowed to sediment onto the surfaceof a suitable filter or membrane at the bottom of a chamber. A samplecan be introduced to the chamber above the cells and pressure applied(by plunger or external pump, for example). As the sample flows past thecells, which are in intimate contact, particles are brought within closerange of the cells, allowing contact.

Counter Flow

One or more layers of cells can be allowed to sediment onto the surfaceof a suitable filter or membrane at the bottom of a ‘cell’ chamber. Asample can be placed in a separate ‘sample’ chamber which is connectedby some flow channel to the cell chamber at a point below the filter.The chambers can be arranged relative to one another such that, in acentrifuge, the sample chamber is closer to the axis of rotation; thelevel of the fluid in the sample chamber being closer to the axis ofrotation than the fluid in the cell chamber. By this means, during therotation of the centrifuge, fluid will flow between the chambers seekinga common distance from the axis of rotation. This can force some of thesample up through the filter supporting the cells and past the cellswhich are being held against that flow by the outward centrifugal force.As the sample flows past the cells, which are in intimate contact,particles are brought within close range of the cells, allowing contact.

Centrifuge Tube Filter

A sample can be introduced to the filter basket of a centrifuge tubefilter with a suitable size cutoff. Under appropriate centrifugeconditions, the sample will be forced through the filter, accumulatingparticles larger than the filter's cutoff size on the surface of thefilter. Cells can be added to the filter basket and be given a briefcentrifugation to bring them onto the filter surface and the particles.

Dielectrophoretic Trap

Similar to the Flow Cell, but with suitable electrodes on any of thesurfaces or projecting into the flow cell. A sample can be introduced bycontinuous flow past the electrodes, which can be connected to andelectrically driven by and external source. For a suitable combinationof flow rate, frequency, waveform, and amplitude, particles can beguided to and captured in a region of minimum electric field intensityabove the cells by negative dielectrophoresis. After stopping the flowand changing the electrical drive to the electrodes (possibly includinga DC voltage on between some electrodes to create an electrophoreticforce), the particle can sediment or be driven (by electrophoresis orpositive dielectrophoresis) onto the attached cells.

Traveling-Wave Dielectrophoresis

In a shallow cylindrical chamber, suitable electrodes (perhapstransparent) can be fabricated on one or both of the parallel faces,including a central planar electrode to collect particles, an electrodearound the periphery, and a set of spiral electrodes (either on the samesurface as the central one or the opposite surface). A sample can beintroduced to the chamber, and a DC potential applied between theperipheral and central electrodes to attract the particles to thecentral electrode by electrophoresis. By an exchange of fluids, cellscan be introduced to the chamber. Energizing the spiral electrodes withthe appropriate phase-shifted AC voltages can sweep the cells to thecenter by traveling-wave dielectrophoresis, where they can sediment ontothe particles.

Dissolvable-Membrane Tube

Use can be made of a electrically-actuated dissolvable gold membrane tomaintain isolation between target particles and emitter cells during thelocalization of the particles by centrifugal sedimentation. Either theparticles can be sedimented onto a membrane over the cells (as shown inFIG. 20), or the cells can be held off from the bottom of the chamber bya membrane spanning the bottom of a separate chamber (perhaps aninsert). In either case, after the membrane has be dissolved byelectrical activation, the particles and cells are mixed bysedimentation, possibly centrifugal.

Acoustics/Ultrasonics

Concentration of particles may be accomplished using acoustic orultrasonic signals. Particles can accumulate at nodes in a sanding wavepattern, or be move by a traveling-wave pattern. Cells can also be movedthis way, or delivered by any of several means discussed above.

Toxin Detection

In order to detect monovalent antigens, it is necessary to inducecrosslinking of surface antibodies using one of two general strategies.First, one can express two independent binding sites on the cellsurface, such that two receptor molecules can bind to a single ligand.Alternatively, one binding site can be expressed on the cell surface ifthe ligand is presented to the cell in a manner in which it appears tobe polyvalent. The following are specific examples using the model ofantibody-antigen recognition.

First, two antibodies can be expressed on the surface of a single cellline, each specific for different epitopes of a individual molecule(epitopes 1 and 2). The binding of a single molecule to two antibodies(one antibody against epitope 1 and another antibody against epitope 2)would initiate crosslinking and light emission. More specifically, asingle B cell line is engineered to express two independent antibodies,each recognizing a different epitope on a single molecule. The presenceof monomeric antigen is now capable of crosslinking the surfaceantibodies, resulting in increased intracellular Ca²⁺ and emission oflight by aequorin. A cell line that expresses functional antibodiesagainst both Y. pestis and F. tularensis (in addition to theendogenously expressed PC antibody) has been tested (see Examples). Eachof these agents is recognized independently by this cell line,indicating that both antibodies are functional and demonstrating thatemittor cells are capable of expressing two functional antibodiessimultaneously.

Another potential issue is the sensitivity of the optoelectronic deviceand methods with an antigen that cannot be pelleted using centrifugalforce. The Yersinia pestis F1 antigen exists as a low molecular weightpolymer in solution, and is therefore not sedimentable in our assay.However, B cells expressing antibody against F1 are capable of detectingsoluble F1 antigen at 5 ng/ml. This compares favorably with currentimmunoassay techniques and demonstrates that the optoelectronic devicecan be quite sensitive to soluble agents. A complementary experiment wascarried out using phosphorylcholine antigen conjugated to ovalbumin. Theability of this small antigen to stimulate antibody crosslinking on thecell surface indicates that this low molecular weight antigen,containing multiple copies of PC epitopes, is able to effectivelycrosslink surface antibodies and generate calcium influx and photonemission.

A second strategy can improve the limit of detection for monovalentantigens shown above by taking advantage of the centrifugal format. Thisapproach utilizes a scheme where one of the toxin antibodies isexpressed on the surface of benign bacteria and the second antibody onthe surface of B cells. The toxin can now be sedimented bycentrifugation, and B cells expressing the second antibody added.Because multiple antigens are immobilized on the surface of thebacteria, the toxin will in essence appear polyvalent to the B cell, andwill initiate a crosslinking event and photon emission. Morespecifically, Antibody against epitope 1 of a monomeric antigen (e.g.toxin) is expressed on the surface of bacteria. Soluble toxin binds tothese antibodies, coating the bacteria with toxin antigen. Thesetoxin-coated bacteria are sedimented by centrifugation prior to additionof B cells expressing antibody against epitope 2. Crosslinking of the Bcell antibodies results in light emission by aequorin. Experimentalresults on this strategy demonstrate the feasibility of detection ofbacterial surface antigens, and the increased sensitivity resulting fromsedimenting those bacteria prior to the addition of B cells. Similarapproaches can also be used for any poorly sedimenting agent to improveits presentation to B cells.

Crosslinking

Crosslinking of target particles can be achieved by any known means. Forexample, crosslinking can be achieved using one or more intermediateagents or molecules such as a peptide, an antibody, a chemical compound,an antibody, biotin, streptavidin, in addition, crosslinking can be viacovalent or non-covalent bonding. Methods for crosslinking also includeprecipitation or attachment to a solid phase via ligands, antibodies orchemical functional groups, as are known in the art.

Multiplexing Assays

The following is a description of how B cell mixtures can be used toincrease the number of detectable antigens without increasing the numberof detection channels (tubes, etc). The simplest way to detect multipleanalytes is to use a single emittor cell type per detection channel andto increase the number of cell assays by increasing the number ofdetection channels. This is acceptable for small numbers of assays but,as increasing numbers of analytes are added, the process becomes morecomplex and resource intensive. It is possible, however, to conduct upto 31 tests with concurrent negative controls in only a 5-channel systemif different B cell lines are mixed together.

As an example, if one has a single channel, one can at most detect asingle B cell assay. If, however, one has two channels, then one candetect 3 separate assays, where each channel contains an equal mixtureof 2 of the 3 separate B cell lines:

For example, if one has 3 B cell lines: A, B, C

And one mixes them into two channels thusly—

2 Channel 1: A, B Channel 2: B, C

Then there are three positive readout possibilities:

Channel 1 Channel 2 Yes No implies only A is present No Yes implies onlyC is present Yes Yes implies only B is present (or that more than oneagent is present, which we will consider unlikely for now)

Similarly, if one has 3 channels, one can detect 7 independent assays,by mixing groups of four cell lines together—

(A convenient shorthand will hereafter be utilized where the cell linesfor individual agents are labeled A through a letter corresponding tothe number of cell lines, and the channel numbers will be written toindicate what channels are required to detect positively for eachindividual agent as follows—123: F—means channels 1, 2, and 3 must allregister positive to ID agent F).

Channel 1 Channel 2 Channel 3 A, B, G, F B, C, E, F C, D, G, F 1: A 12:B 123: F 2: E 13: G 3: D 23: C

A formula embodying the relationship that simply describes the number ofindependent assays that can be accessed by a given number of channels,assuming all assays are mixed in equal proportion is:

#Cell assays=2^(n)−1 where n is the number of channels

and the number of cell assays that need to be mixed in each channel isgiven by 2^((n−1)).

Thus, to mix 16 different B cell lines together, 5 channels are neededto interrogate 31 different assays. The design for a 10-channel systemcould, in fact, be used to provide ID for 31 separate agents withconcurrent negative controls (5-channel positive ID, 5-channel negativecontrol).

The channel mixtures and positive detection correlation for a 4-channelsystem (15 different assays) is shown below:

Channel 1 Channel 2 Channel 3 Channel 4 A, B, G, F, B, C, H, I F, C, D,I D, E, G, H I, K, L, M J, L, M, N J, K, M, O J, K, L, M 1: A 23: C 123:I 1234: M 2: N 24: H 234: J 3: O 34: D 134: K 4: E 12: B 124: L 13: F14: G

Without further elaboration, it is believed that one skilled in the artcan, based on the above disclosure and the examples below, utilize thepresent invention to its fullest extent. The following examples are tobe construed as merely illustrative of how one skilled in the art canpractice the invention, and are not limitative of the remainder of thedisclosure in any way.

EXAMPLES

FIG. 1 is a schematic diagram showing the general cellular components ofthe invention. A cell (here a B cell) that contains an emitter molecule(here aequorin) has antibodies present on its surface. These antibodiesare specific for an antigen on a target particle, such as a biologicalwarfare agent. Binding of the target particle to antibodies on the Bcell brings two or more antibodies close together on the cell surface,causing a signal transduction cascade that leads to release of calciumfrom intracellular stores into the cytoplasm. This increase incytoplasmic calcium concentration causes aequorin to emit a photon. Thephoton is then captured and registered by a photo multiplier device,such as a CCD. Thus, a cellular biosensor can be implemented using cellshaving functional surface antibodies and containing a cytoplasmicemitter molecule that responds to increased calcium concentration.

Such a cell-based detection system provides rapid, sensitive, specific,accurate, and flexible detection of any antigen on any target particle.In regard to flexibility, the system can be modified to target anyparticle or groups of particles. In one example, a single emitter cellcan contain a plurality of antibody types, each type being specific fornon-overlapping groups of target particles. This single emitter cell canthen be used to identify a genus of target particle species at once.

In a second example, a reaction chamber can contain two types of emittercells. One type of emitter cell contains antibodies that are specificfor a genus of target particles (e.g., bacteria) and emits a photon of afirst wavelength in response to contact with any member of the genus.The second type of emitter cell contains antibodies that are specificfor a particular species within the genus (e.g., Yersinia pestis) andemits a photon of a second wavelength different from the firstwavelength in response to contact with the species. This arrangementoffers extremely high accuracy by reducing or eliminating false positivesignals. Only when photons of the first and second wavelength aredetected, would a positive event be registered. This nesting of emittercell specificities can be extended to more than two levels as necessaryto reduce or eliminate false positive signals.

FIG. 2 is a schematic diagram of a general architecture and useenvironment for the invention.

FIG. 3 is a schematic diagram of the molecular biology employed in oneembodiment of the invention. In this example, a universal B cell linethat expresses an emitter molecule (e.g., aequorin) but does not expressantibodies becomes the basis for generating B cells that can express anyantibody specific for any antigen. An antibody expression vector isintroduced into the universal B cell, selected for the presence of theexpression vector, and expanded for use in a detection system of theinvention. Using this strategy, in conjunction with pDisplay andVKExpress (described in “Antibodies” section above), target-specificemitter cells were generated for a variety of targets. Emitter cellsspecific for Foot and Mouth Disease virus (FMDV), Venezuelan EquineEncephalitis (VEE) virus, Yersinia pestis, Francisella tularensis,Brucella spp., the O1 and O139 strains of Vibrio cholera, and orthopoxviruses have been produced. The cDNA and sequence for the FMDV antibodyvariable regions were obtained from the USDA. The cDNAs and sequencesfor the Yersinia pestis, Francisella tularensis, Brucella spp., the O1and O139 strains of Vibrio cholera antibody variable regions wereobtained from investigators at NMRC. The variable regions of the VEE andorthopox antibodies were cloned from hybridomas obtained from the CDCand USAMRIID, respectively. Foot and Mouth Disease Virus (FMDV),Yersinia pestis, Francisella tularensis, and Venezuelan EquineEncephalitis Virus (VEEV) are responsible for Foot and Mouth Disease,the Plague, tularemia, and encephalitis, respectively. Cloning from thehybridomas was done with a combination of primers described in severalpublished articles. Emitter cells specific for Bacillus globigii arebeing produced because this non-pathogenic bacterium is used by somemilitary agencies as a test organism in field trials of biologicalwarfare agent detection systems. FIG. 5 includes a line graph showingthe photon emission response when several clones of FMDV-specificemitter cells were contacted with live FMDV targets. In each case, theemitter cells fired photons within about 20-30 seconds after contactbetween the target and the cells. Included in the graph is data showinga lack of emission when a mutant FMDV (having single amino acid mutationin the viral coat protein) that would not be expected to bind to theemitter cell was contacted with an emitter cell clone. The negativecontrol supports the high specificity that is built into the detectionsystem.

Various configurations of a centrifuge and photomultiplier tube (PMT)arrangement can be incorporated into a system of the invention. Thearrangement includes a rotor (motor) that spins a sample microfuge tubefrom a swinging harness and includes a balance tube in a fixed position.The PMT is shown at the bottom, facing upwards toward the bottom end ofsample tube at rest. In a typical experiment for a target particle thatis smaller than the emitter cell, the particle-containing liquid sampleis placed in the sample tube and centrifuged under conditions sufficientto sediment the majority of the particles to the bottom of the tube(e.g., 60 seconds at 5600×g for Francisella tularensis). A suspension ofemitter cells is then layered onto the sample in the tube (so as not todisturb the sedimented particles) and spun briefly to pellet the cellsinto contact with the target particles. If target particles are presentin the candidate particles, photons of a specific wavelength should beemitted from the cells and captured and registered by the PMT.

In specific embodiments, the PMT can be a Hamamatsu HC 125-08 PMTinterfaced with a Stanford Research systems SR400 Two Channel GatedPhoton Counter. The centrifuge can be a Sapphire 17 turn, 18.5 AWG, 5amp motor having a swinging bucket configuration.

The centrifuge tube (reaction chamber) can be altered and upgraded asneeded to aid contact between candidate particles and the emitter cells.In one embodiment shown in FIG. 20, the tube contains an enclosedcompartment that holds pre-loaded emitter cells at the bottom of thetube. This compartment is separated from the rest of the tube by adissolvable gold anode membrane. In operation, a same containingcandidate particles is deposited into the tube and spun to concentratecandidate particles at the membrane. The membrane is then dissolved, andthe tube briefly spun to contact the particles with the emitter cells.This dissolvable membrane system is described by Langer and colleaguesin Angewantde Chimie International Edition 39:2396-2407, 2000; andNature 397:335-338, 1999.

The steps in the centrifuge process can be automated or alternativelydesigned so that the user need not stop the centrifuge at all. Forexample, the introduction and removal of liquids and samples can beaccomplished without the need to stop the rotor by adapting themechanical features of preparative centrifuges (e.g., ultracentrifuges)available from Beckman Instruments. In addition, it may be desirable todetect photon emission while centripetal forces are still being applied(e.g., when the contact between emitter cells and target particles areunstable without centrifugation). To detect photons emitted from thesample tube while it is spinning, the PMT can be arranged in a radialposition relative to the rotor axis. In most cases, the PMT in thisarrangement need not be spinning along with the sample tube, but insteadcan be stationary and simply register emission of photons when thesample tube passes in front of the PMT. If the emission signal is veryweak, then the detector (e.g., PMT, a CCD chip) can be coupled to therotor and spun along with the sample tube. Alternatively, multiple PMrscan be arrayed around a circumference of a rotor for detectingemissions.

If multiple samples are spun on the same rotor, the positioning orsignal processing of the PMT can be altered if necessary. In oneembodiment, the rotor accommodates 4 sample tubes, each containing cellsthat emit at the same wavelength. To differentiate emissions originatingfrom one sample over the emissions from another, a single radiallyaligned PMT can detect emissions continuously. The continuous emissiondata is then resolved using a timing trace from the rotor, whichmonitors the position of each sample over time, to allocate theemissions to each sample. Other variations are understood to be withinthe invention.

For example, FIG. 17 is a schematic drawing of two reaction tubescoupled to a rotor, with two PMTs aligned below the tubes. At a restingposition, the rotor positions each of the tubes below the correspondingPMT, using the rotor position encoder. In another example, thecentrifuge system shown in FIG. 17 can be integrated with an air samplecollector to achieve the system shown in FIG. 18. The radial aerosolimpactor tube can include any type of particle monitor, such asdescribed in U.S. Pat. No. 5,932,795 and references cited therein. Instill another example, the system described in FIG. 18 can be altered sothat only one PMT aligned radially in relation to the rotor axis isrequired, as shown in FIG. 19. As discussed above, emissions registeredby the PMT are resolved for each sample tube using the shaft encoder.

Referring back to FIG. 17, fluid components including, but not limitedto, suspensions of B cells engineered to recognize specific bioagents,buffer solutions, preservatives, cell culture medium, can be placed ineach of several centrifuge tubes, mixed with a liquid suspension of thesample particles that has previously been collected from aerosol samplesin a separate process particles may include but are not limited to,proteins, peptides, chemicals, viruses, bacteria in vegetative and sporeforms, fungal spores, pollen grains, protozoa, blood or tissue derivedcells, and fragments thereof either alone or in conjunction with carrierparticles such as dust). When the spin motor is started, the centrifugetubes swing out into a radial position, and the B cells and/or sampleparticles are driven to the bottom of the centrifuge tubes at ratesdepending upon the size and density of the particles. The exact sequencewhereby cell and sample-containing fluids are added and centrifuged canbe customized based on their relative sedimentation velocities tomaximize signal. In general, it is expected that maximum photon outputcan be obtained from particles that sediment more slowly than B cells byspinning these samples (a pre-spin) for appropriate times before theaddition of B cells and spinning to bring them into contact. Forparticles sedimenting at similar or faster rates than B cells, a singlespin of the mixed sample and B cell components will initiate maximalphoton output from the system. Data from particle size analyzers(including but not limited to BAWS units, and fluid particle analyzers)incorporated upstream of the centrifugation device can be used toautomatically determine the optimal operation sequence and initiateappropriate computer-controlled automated sample handling.

When the “spin cycle” is terminated and the rotor comes to a controlledstop in a pre-determined position controlled by the spin motor and shaftencoder, the swing arms rotate under gravity forces so that the bottomsof the centrifuge tubes are presented to the sensitive surface of thephotomultiplier tubes, and any light signals are then recorded. In amodified version of this implementation, a single photomultiplier tubecan be positioned at the maximum radius of the rotor/tube configurationand used to collect photons from each tube as they pass by the sensitivesurface of the photomultiplier tube in succession. The photon outputmeasured from individual tubes can be assigned and combined based on themonitoring of the shaft encoding system.

Referring back to FIG. 18, the process of collection of the aerosolparticles is integrated with the process of bringing the aerosolparticles into contact with the B cells. Here, the centrifuge tubes areattached to swing arms that allow them to cover the ends of radialimpactor tubes while spinning, and the aerosol sample is induced to flowinto the sample inlet by the centrifugal forces acting on the air in therotating radial impactor tubes (can be assisted as necessary byadditional blower units). The high velocity of the flow causes aerosolparticles to impact on the inner surface of the centrifuge tube or thesurface of a liquid contained in the tubes and results in the capture ofthe particles on the surface of the tube or in the liquid, respectively.When a liquid is present, centrifugal pressures acting on the liquidwill balance the force imparted by the high velocity air flow requiredfor particle capture in the liquid and prevent it from being blown outby the impacting air. The aerosol particles are retained followingimpact with the tube surface or liquid and in the case of liquidcollection, forced to flow radially outward thereby providing increasedlocal particle concentrations at the maximum radius (the bottom of thecentrifuge tube). Addition of B cells and spinning them into the locallyconcentrated particle zone following the collection phase will initiatephoton output. Alternatively, the B cells can be present in the fluidduring collection and light output monitored in real time while spinningwith a single photomultiplier tube (FIG. 19). In a modified version ofthis implementation, the fluid components (including but not limited toparticle samples collected via an alternative bioaerosol collector, andsuspensions of engineered B cells) could be added to the inlet(s), andthe centrifugal forces can be used to distribute them to the tubes.

When the “spin cycle” is terminated and the rotor comes to a controlledstop in a pre-determined position controlled by the spin motor and shaftencoder, the swing arms rotate under gravity forces so that the bottomsof the centrifuge tubes are presented to the sensitive surface of thephoto multiplier tubes, and any light signals are then recorded. In amodified version of this implementation, a single photomultiplier tubecan be positioned at the maximum radius of the rotor/tube configurationand used to collect photons from each tube as they pass by the sensitivesurface of the photomultiplier tube in succession. The photon outputmeasured from individual tubes can be assigned and combined based on themonitoring of the shaft encoding system.

FIG. 7 is a schematic representation of the results of sequentialcentrifugations. For target particles smaller than emitter cells buthaving the same density of emitter cells, it is beneficial to first spinthe candidate particles (e.g., at high speed) to pellet them. Thereafterthe emitter cells can be added and spun under conditions which can bemilder to prevent reduction of their responsiveness as needed (topseries). In addition, this sequence of centrifugation forces almost allcandidate particles and emitter cells into a relatively small volume atthe bottom of a centrifuge tube. In contrast, mixing the candidateparticles and the emitter cells together and spinning them at one timewill lead to separation rather than contact between the particles andemitter cells (middle series). Of course, no spin at all dramaticallyreduces the effective concentration of particles and emitter cells inthe reaction (bottom series).

FIG. 8 includes a line graph showing in an actual experiment confirmingthe consequences proposed in FIG. 7. Emitter cells specific forFrancisella tularensis were mixed with killed Francisella tularensiscells in the three methods shown in FIG. 7. As seen in the line graph,the sequential spin method resulted in fast and efficient emission aftercontact. In contrast, the emission profile of the single spin method wasfar less pronounced in both timing and magnitude. The no-spin methodbarely exhibited a reaction.

A similar emission profile was generated in a separate experiment, assummarized in the line graph shown in FIG. 8. Inspection of the emissiontraces suggested that the single spin method resulted in target-specificemissions a little quicker than the two-spin method. However, thisresult was found to be primarily an artifact of the longer spin requiredfor the two-spin method and does not reflect an actual improvement inthe response time of the B cells. In fact, the initial slope of thetwo-spin method was significantly greater than that for the single spinmethod, indicating that the two-spin method led to a robust emitterresponse.

The sensitivity of the detection system shown in FIG. 8 was evaluated bytitrating the number of tularemia cells deposited into the centrifugetube. The results are summarized in the line graph shown in FIG. 10. Itappears that 25,000 emitter cells were capable of emitting photonsdetectable above background in response to about 5,300 tularemia targetparticles. It is expected that further optimization of reactionconditions will increase sensitivity.

Cell responses are improved after a single freeze-thaw cycle (see FIG.22). In this experiment, cells specific for Yersenia pestis (YP) werecentrifuged, resuspended in freezing medium (RPMI with 10% DMSO and anadditional 10% FBS), frozen at −80° C., and transferred to liquidnitrogen. Cells were thawed at 37° C. and 1 ml (2×10⁶) cells werediluted into to 4 mls of RPMI and incubated overnight at 37° C. Thefollowing day the cells were loaded with coelenterazine for 2 hours,washed into CO₂—Independent medium (CO₂—I) and recovered for 24 hours.10,000 cells were challenged with 5×10⁵ YP (50 ul of YP at 10⁷/ml).Untreated cells displayed a response of 9500 photons per second, whilefrozen thawed cells emitted approximately 6 fold more photons inresponse to YP. This stimulatory effect could be largely replicated byexposing the cells to freezing medium, without the actual freezing (5fold stimulation). It appears that the stimulatory factor in thefreezing medium is the DMSO. When cells were treated with 2% DMSO (thefinal concentration of DMSO when 1 ml of cells in freezing mediumcontaining 10% DMSO is diluted into 4 mls of normal medium) a similarlevel of stimulation was detected. The DMSO effect may be due to anumber of factors. DMSO is known to effect hematopoetic celldifferentiation, and may be stimulating the cells through this pathway.Additionally, cells frozen in medium containing glycerol also showsimilar levels of stimulation. Thus, it appears that the effect can alsoin part be due to a stress response induced by the DMSO and it can bepossible to replicate this stimulation using any of a number ofconditions that stimulate a “heat shock” response.

The cells can be stored frozen in the coelenterazine-charged state.Cells were loaded with coelenterazine, allowed to recover for 24 hours,and then frozen. Upon thawing the cells were washed through 10 ml ofCO₂—I medium and the cells were resuspended in CO2I medium to aconcentration of 5×10⁵ cells/ml. These cells were capable of detectingYP (in this case about 1 hour after thawing, but shorter times arepossible). These cells remained capable of detecting agent for severaldays when stored at RT. Pretreatment of these cells with DMSO, prior toloading with coelenterazine and freezing, can increase the sensitivityof the cells to agent after thawing.

In FIG. 22, cells were challenged with 50 ul of 10,000,000 YP/ml dilutedin CO₂—I after various cell treatments. Untreated: Cells were grown inRPMI, loaded with coelenterazine, washed, recovered for 24 hours, andchallenged with YP. Freeze/Thaw: Cells were grown in RPMI, transferredto freezing medium, and frozen. Thawed cells (1 ml) were placed into 4mls of RPMI and incubated at 37° C. for 24 hours, loaded withcoelenterazine, washed, recovered for 24 hours, and challenged. FreezingMedium: Cells were grown in RPMI, transferred to freezing medium andincubated at RT for 10 minutes. Cells (1 ml) were placed into 4 mls ofRPMI and incubated at 37° C. for 24 hours, loaded with coelenterazine,washed, recovered for 24 hours, and challenged. 2% DMSO: Cells weregrown in RPMI, transferred to RPMI containing 2% DMSO and incubated at37° C. for 24 hours, loaded with coelenterazine, washed, recovered for24 hours, and challenged.

A successful biological warfare detection system should be resistant tocontamination by common environmental substances present on abattlefield. To evaluate whether emitter cells can operate underenvironmental stress or contamination, emitter cells were mixed with atarget particle after exposure of the emitter cells to one hour of fullstrength diesel exhaust (left line graph in FIG. 11), or when theemitter cells were contaminated by 10⁷ E. coli (right line graph in FIG.11), which was used as a surrogate contaminant for any field bacterium.As shown in FIG. 11, the particular chemical and biological contaminantstested did not affect the ability of emitter cells to fire photons inresponse to a target particle.

FIGS. 13-14 describe a different embodiment of the invention that doesnot require centrifugation. The schematic diagram of FIG. 13 shows thevarious components of this embodiment. A biological aerosol warningsensor (BAWS) detects the present of particles, e.g., within apre-determined size range. An example of a BAWS is described inPrimmerman, Lincoln Laboratory Journal 12:3-32, 2000. If particlesmeeting specifications are detected, BAWS triggers an air-to-airconcentrator (specimen collector; as described in U.S. Pat. No.5,932,795) that allows particles of a particular size range to becollected and deposited in a well (reaction chamber) on a portion of aspecimen tape at a first station, which is shown in different views inFIG. 14. After candidate particles are deposited in the well, the tapeadvances to a second station under a reservoir of emitter cells and overa PMT. Emitter cells specific for a particular antigen on a targetparticles are deposited in the well, and the photon emission from thewell monitored.

During the time that candidate particles are detected by BAWS, thecandidate particles can be deposited on consecutive wells as the tape isadvanced through the first station (FIG. 14). In the second station, aplurality of emitter cell reservoirs, each containing emitter cellshaving different target specificities, are mounted on a turret thatrotates a particular reservoir into position to deposit differentemitter cells into the well. In this manner, different targets withinthe candidate particles can be detected, as shown in the schematic topview of the wells in FIG. 14. Of course, if the different emitter cellsemit at different wavelengths, it is possible to deposit the differentemitter cells into a single well containing candidate particles,provided that the PMT below the second station can distinguish photonsof different wavelengths.

FIG. 16 shows schematically yet another embodiment of a system of theinvention. In this embodiment, air particles are deposited in each ofsix wells within a row of a two-dimensional array (e.g., a tape having 6rows and hundreds of columns) at a first station. As the array isadvanced by one row, positioning the row in a second station, differentemitter cells are deposited into each well within the row, and emissionfrom all six reactions is detected simultaneously by a row PMTs underthe second station. To aid adhesion of particles to the wells on thetape, the wells can be coated with an adhesive or a liquid.

CELL ENGINEERING AND ASSAY METHOD EXAMPLES A. Cell Engineering Methods

M12g3R cells were maintained at 37° C. in a humidified atmosphere of 5%CO₂ in RPMI 1640 supplemented with 10% fetal bovine serum, 1 mM sodiumpyruvate, 2 mM L-glutamine, 100 μM nonessential amino acids, 50 μM2-mercaptoethanol, 50 μg/ml streptomycin, and 50 U/ml penicillin, 250ng/ml amphotericin B (Life Technologies). Cells were transfected withpCMV.AEQ.IRES.NEO via electroporation (270 V, 950 pF) and selected in 1mg/ml G418 for two weeks. G418-resistant clones were screened forresponse to anti-IgM. Those clones with the greatest increase in photonemission upon crosslinking of the surface IgM were used in subsequenttransfections to generate B cell lines specific for particularpathogens. Surface expression of antibodies with engineered specificityis accomplished by co-transfection (via electroporation) with expressionvectors for light and heavy chains, as well as with one that encodes agene conferring resistance to puromycin. Puromycin-resistant pools andclones were selected on the basis of their response to antigen. Thelight chain expression vector, VKExpress, contains the constant regionfor the human kappa gene downstream of a multiple cloning site (MCS),under control of the human elongation factor-1α (EF-1α) promoter. Theheavy chain vector was generated by modifying pDisplay (Invitrogen),retaining the cytomegalovirus (CMV) promoter and leader sequence, butreplacing the platelet-derived growth factor (PDGF) receptortransmembrane domain with the gene for the membrane-bound constantregion of murine IgM and removing both tags on either side of the MCS.The appropriate restriction sites are added to the antibody variableregions using PCR and the sequence of all PCR products is confirmedbefore cloning into the expression construct. The variable regions usedto produce the recombinant antibody were cloned either from cDNA or fromhybridomas using Reverse-Transcription (RT) with random oligonucleotideprimers and PCR. RNA was extracted with Trizol reagent (LifeTechnologies), according to the manufacturers recommendations, and firststrand synthesis performed using the Retroscript kit (Ambion). PCR wasaccomplished using sets of primers designed to anneal to the leadersequences of either light or heavy chains [S. T. Jones and M. M. Bendig,Bio/Technology 9, 88 (1991)] at the 5′ end, and the constant regions ofmurine Kappa or IgG2 at the 3′ end.

B. Bioluminescent B Cell Response to FMDV

The M12g3R B cell line, stably transfected with the pCMV.AEQ.IRES.NEOplasmid and expression vectors for a recombinant antibody thatrecognizes the A12 strain of FMDV, was prepared for the luminesenceassay as follows: Cells were thawed on Day 1. Preparation of the cells24 hours post-thaw is critical for maximum activity and reliability. Thefreeze/thaw step increases the response of the B cells by as much as 100fold. On Day 2, 10⁶ cells were incubated at room temperature for 2 hoursin assay medium [CO₂—Independent medium, 10% FBS, 50-μg/ml streptomycin,and 50-U/mil penicillin, 250 ng/ml amphotericin B (Life Technologies)]with 50-μM coelenterazine (Molecular Probes, Eugene, Oreg.) covered withfoil, washed twice, and resuspended in assay medium at a finalconcentration of 5×10⁵ cells/ml. Cells were left rotating overnight atroom temperature in 1.5 ml microcentrifuge tubes and assayed 15-20 hourslater. For the assay, 25 μl of cells was mixed with antigen (5 μl of thewt A12pRMC35 strain at 1.4×10⁸ pfu/ml, 10 μl of the A12 variant, B2PD.3,at 7.5×10⁷ pfu/ml) and the response measured in a luminometer (Lumat LB9507, Perkin Elmer).

C. Bioluminescent Assay with Bacteria and Large Viruses

The sensor device and methods can be used for the rapid detection ofbacterial, as well as viral pathogens. Cell lines were engineered torespond to the bacterium, Francisella tularensis, the etiological agentof tularemia. However, when assayed using the same protocol as with theFMD and VEE viruses, the signal is slow and almost indistinguishablefrom background, indicative of low interaction rates between the B cellsand antigen (0 s pre-spin/0 s spin). Previous experiments performed withantigen-bead simulants have indicated that sensitivity and speed couldbe augmented by concentration of antigen and B cells (data not shown),so the luminometer was re-configured to include a centrifuge positionedabove the photomultiplier tube (PMT). When the agent and cells are mixedtogether, then concentrated by centrifugation for 5 seconds, the signalis improved and the response faster (0 s pre-spin/5s spin). Optimalresults are observed when the slower-sedimenting F. tularensis iscentrifuged prior to the addition of the cells (60 s pre-spin/5s spin).This format ensures that a large number of cells come into physicalcontact with antigen within a short time frame, thereby providing amajor improvement in sensitivity and speed. After additionaloptimization of the assay protocol, we can now detect as little as 60colony-forming units (cfu) of F. tularensis in less than 3 minutes,including the time it takes to pre-spin the agent, but see no responseto inactivated Yersinia pestis, the bacterium that causes the plague.This limit of detection has been confirmed with two other sources ofinactivated F. tularensis, and one different strain (data not shown).Furthermore, the sensor device exhibits a wide range of sensitivity,detecting concentrations ranging over 7 orders of magnitude.

B cells were prepared as described above. 50 μl containing the indicatedamounts of Y. pestis or F. tularensis were centrifuged for 60 s at6500×g, then 20 μl of cells were added and spun an additional 5 s in thecentrifuge luminometer. Photons were detected with a Hamamatsu HC-125photomultiplier tube and the signal monitored with a Stanford ResearchSystems SR400 Gated Photon Counter.

NUCLEIC ACID DETECTION EXAMPLE Characterization of Emittor CellsExpressing Digoxigenin Antibody

Plasmids encoding an antibody (Daugherty et al. (1998) ProteinEngineering 11 (9): 825-832) against digoxigenin were introduced intoemittor cells, and these cells were screened using protein (BSA)chemically conjugated to digoxigenin (Dig-BSA). Twelve independent poolswere selected resulting in 12-24 independent cell lines. The firstexperiment tested whether these cells could detect digoxigenin antigenscrosslinked by DNA (Dig-DNA). Three types of commercial Dig-DNA havebeen tested for reactivity with Dig antibody expressing CANARY cells(FIGS. 26A-C): plasmid DNA with a digoxigenin attached every 20 basepairs (FIG. 26A); DNA molecular-weight markers with digoxigenin attachedevery 200 bases (FIG. 26B); and DNA molecular-weight markers with onedigoxigenin attached to each end (FIG. 26C). Each of these standardsstimulated the emittor cells to a varying degree, with the mostsensitive response being to the Dig-labeled plasmid DNA. The fact thatantigens spaced an average of 20 bases apart stimulate the cells 100fold more (on a per digoxigenin basis, not on a per microgram of DNAbasis) than antigens spaced 200 bases apart is an indication that 200bases is too great of a distance to stimulate an ideal response. Inorder to stimulate an intracellular cascade resulting in calcium releaseand aequorin light production, adjacent antibodies must be immobilizednear enough to each other to initiate the reaction inside the cell.

It was also noted that centrifugation just before measurement of lightoutput, which is routine in the detection of both soluble and insolubleantigens using traditional CANARY, may actually decrease the sensitivityof CANARY against the soluble Dig-DNA antigen. In the experiment shown(FIGS. 27A and 27B), centrifuging the cells through the DNA solutionappears to decrease the limit of detection by nearly a factor of 10.This result may reflect differences between detection of DNA anddetection of other nonsedimentable antigens.

Detection of Hybridized Oligonucleotide Probes Using Emittor Cells

This assay was designed to detect hybridization of digoxigenin-labeled(Dig-labeled) probes to target DNA. The target DNA for these experimentswas derived from the phagemid pBluescript II. This 3100 base pair-longcircular phagemid can be induced to make double-stranded DNA (dsDNA) oreither of the two single strands of DNA (ssDNA). These two ssDNA strandsare termed the (+) strand or the (−) strand. Ten Dig-labeledoligonucleotide probes that bind specifically to the (+) strand weredesigned:

Oligo Phagemid number DNA Sequence base position Tm  1 GCAACGTTGTTGCCATT(SEQ ID NO: 1) 2269-2285 56.0  2 TACAGGCATCGTGGTGT (SEQ ID NO: 2)2288-2304 53.3  3 GCTCGTCGTTTGGTATGG (SEQ ID NO: 3) 2309-2326 57.3  4TCATTCAGCTCCGGTTC (SEQ ID NO: 4) 2328-2344 55.0  5 ACGATCAAGGCGAGTTAC(SEQ ID NO: 5) 2348-2365 53.1  6 GATCCCCCATGTTGTGC (SEQ ID NO: 6)2368-2384 57.7  7 AAAGCGGTTAGCTCCTTC (SEQ ID NO: 7) 2388-2405 54.3  8TCCTCCGATCGTTGTCA (SEQ ID NO: 8) 2408-2424 56.5  9 GTAAGTTGGCCGCAGTG(SEQ ID NO: 9) 2428-2444 55.7 10 TCACTCATGGTTATGGCA (SEQ ID NO: 10)2448-2465 53.5 NEG3 CCATACCAAACGACGAGC (SEQ ID NO: 11) 2326-2309 57.3Oligonucleotides are numbered in the order of their location along thepBluescript phagemid DNA. Shown for each is the DNA sequence of theoligonucleotide, the position of that sequence on the phagemid, and themelting temperature (Tm) of that oligonucleotide (an approximation ofthe binding affinity). The small range in Tm's for theseoligonucleotides indicate that they each have similar bindingcharacteristics.

Each of these oligonucleotides has a digoxigenin (Dig) molecule attachedto the first residue, and each have comparable target DNA bindingcharacteristics as reflected by their similar calculated meltingtemperatures (Tm). Hybridization of this set of 10 digoxigenin-labeledoligonucleotides to the (+) strand of the target DNA yields a 200 basestretch of double-stranded DNA with one Dig molecule every 20 bases. Theremaining 2900 bases of the plasmid remain single stranded. Thiscollection of immobilized digoxigenin antigens crosslink digoxigeninantibodies on the surface of emittor cells and stimulate lightproduction.

The 11th oligonucleotide (NEG 3) is a control. NEG 3 was designed tobind directly to oligonucleotide number 3, producing a short piece ofdsDNA 20 nucleotides long with a single Dig on each end. Emittor cellsexpressing a digoxigenin antibody were capable of detecting 80femptomoles of input oligonucleotide (FIG. 28). This controldemonstrated that the hybridization conditions chosen were at leastsufficient to support binding of two oligonucleotides within this Tmrange. More importantly, this control demonstrated that the bindingbetween 20 bases of complementary DNA is sufficiently strong tocrosslink antibodies and elicit a signal from the emittor cell.

Oligonucleotide-oligonucleotide hybridization occurs extremely quickly(FIG. 29). Oligonucleotide NEG3 was added to hybridization solution,followed by Oligo3. The solution was immediately diluted in medium, theemittor cells added, and the reaction place in the luminometer (elapsedtime from addition of oligo 3 was 15 seconds). This abbreviatedhybridization protocol did not drastically affect the sensitivity of theassay (compare FIG. 29 to FIG. 28).

Next, multiple Dig-labeled oligonucleotides were hybridized tosingle-stranded DNA target. This complex was tested for its ability tostimulate emittor cells. FIG. 30 shows a series of hybridizations ofdifferent concentrations of the Dig-oligonucleotide probe set to a givenamount of ssDNA. The ratio of ssDNA:oligonucleotide probe giving thebest signal in this experiment was between 1:2 and 1:4. At higherconcentrations of probe, the unbound Dig-labeled oligonucleotideappeared to inhibit signaling. In these experiments 0.63 pmoles ofoligonucleotide worked well under many of the conditions tested. Adose-response curve gives a limit of detection for single stranded DNAof approximately 50 ng, or about 50 fmoles (FIG. 31). It is important tonote that (−) strand DNA was not detected in either of theseexperiments, indicating hybridization of the Dig-labeledoligonucleotides and subsequent signaling from the emittor cells isdependent on the sequence of the target DNA.

Temperature and buffer constituents affect hybridization of Dig-oligosto target NA. Hybridization at between 47° C. and 51° C. in either PBS(FIG. 32A) or TBS (FIG. 32B) gave the highest response. Hybridizationsperformed at higher or lower temperatures decreases the amplitude of thesignal. Changes in the buffer constituents will obviously affect theideal hybridization temperature.

Target DNA Capture

Biotin-labeled oligonucleotides have been bound to the surface ofstreptavidin-coated magnetic and nonmagnetic beads. These “capture”oligos are designed to bind to the target DNA in a position well removedfrom the location of the Dig-labeled oligonucleotides. Binding thetarget NA to a sedimentable support will allow for more extensivewashing of the DNA before addition of emittor cells, and improve thesensitivity of the assay. One strategy for sedimentation of target NA isshown in FIG. 33. In this scheme, a biotin-labeled captureoligonucleotide is attached either streptavidin-coated polystyrene ormagnetic beads. Digoxigenin-labeled oligonucleotides are hybridized tothe target, and the complex sedimented by centrifugation or applicationof a magnetic field. The emittor cells are then resuspended andsedimented with the beads, and the reaction tube placed in aluminometer. The effects of sedimentation on detection of target DNA isshown in FIG. 34. In this case, the LOD is improved to the high attomolerange as compared to typical results in which the DNA is not sedimented.The addition of a commercial blocking reagent (Roche Applied ScienceCat. # 1 096 176) improves signal further. FIG. 35 shows the result ofaddition of different concentrations of blocking agent during thehybridization/capture step. In this experiment, addition of between 1%and 10% blocking reagent improved the signal to background ratio at allconcentrations of target tested.

Fc Receptor Emittor Cells

The Fc receptors are a family of membrane-expressed proteins that bindto antibodies or immune complexes. They are expressed on severalhematopoietic cells including monocytes and macrophages. Severalsubclasses of Fc receptors exist including Fc gamma Receptor I (FcγRI),a high-affinity binder of soluble antibody. FcγRI binds to the constantregion (Fc portion) of Immunoglobulin G (IgG) leaving theantigen-binding region of the antibody free. Crosslinking of theantibody-bound receptor by specific antigen initiates a signalingpathway that stimulates calcium release.

The human macrophage cell line, U937, contains endogenous FCγRI.Treatment of these cells with IFNγ increases the expression of theFcγRI, as seen in FIG. 36A. U937 cells transfected with the aequorinexpression plasmid produce functional aequorin as demonstrated bytreating these cells with the calcium ionophore ionomycin. This causes arapid and transient rise in calcium that stimulates the aequorin to emitlight, as seen in FIG. 36B. U937 cells were then tested to determine ifthe aequorin would be stimulated by the calcium rise initiated bycrosslinking of the Fc receptors. U937 cells were incubated with humanIgG for 15 min at room temperature. The cells were washed to removeunbound IgG and treated with goat anti-human IgG. A rapid rise incalcium was observed, as shown in FIG. 36C.

Experiments demonstrated that U937 cells can be “engineered” rapidly torespond to several different pathogens or simulants. U937 cells weretreated for 24 h with IFN (200 ng/ml) to increase expression ofendogenous FcγRI, and prepared for the emittor cell assay. The cellswere incubated with the following antibodies: mouse anti-B. anthracisspore (FIG. 37A), rabbit polyclonal anti-B. anthracis spore (FIG. 37B),mouse anti-F. tularensis (FIG. 37C), or mouse anti-B. subtilis (FIG.37D). Cells were then used in the standard assay where they detected asfew as 1000 cfu B. anthracis spores with the monoclonal antibody and10,000 cfu spores with the rabbit polyclonal, as well as 10,000 cfu F.tularensis and 1,000 cfu B. subtilis spores.

The next set of experiments demonstrated that the specificity of theassay is determined by the antibody that is used. U937 cells wereincubated with mouse anti-F tularensis antibodies and were tested fortheir response to 105 cfu of B. anthracis spores. As shown in FIG. 38A,the cells did not respond to B. anthracis but did to 106 cfu of F.tularensis. Alternatively, cells loaded with mouse anti-B. anthracisspore antibodies did not respond to F. tularensis but did to 106 cfu ofB. anthracis spores, as shown in FIG. 38B. Furthermore, the cells didnot show any response to the 106 cfu of F. tularensis in the absence ofanti-F. tularensis antibody, as seen in FIG. 38C.

16 Channel Sensor Example

A new approach that reduces the time to measure multiple samples (whilekeeping the hardware requirements minimal) has been successfully tested.An experimental sensor has been designed that allows the simultaneousmeasurement of 16 samples using a single light-gathering channel. Thesensor consists of a rotor holding sixteen 1.5-ml tubes horizontally,equally distributed about its circumference, and driven by a variablespeed motor about a vertical axis (FIG. 39). A single fixedphoton-detecting element (in this case, a PMT) is positioned in theplane of the rotor just beyond the path of the tubes during rotation. Inthis way, each of the tubes is sequentially and repetitively broughtinto close proximity to the PMT, allowing its light output to be sampledon each pass. Finally, an optical switch consisting of an optical source(an infrared LED) and a detector (a phototransistor) is used to controlthe counting of detected photons and the reorganization of the data into16 fields, each associated with a specific sample.

A single measurement consists of:

1. Preparing 16 samples (and/or controls) in individual 1.5-ml tubes;2. Introducing an aliquot of emittor cells into each of the tubes;3. Installing the tubes into the rotor situated in a dark box;4. Localizing the emittor cells at the bottom of the tubes using a brief(5 sec) centrifugal spin at high RCF (˜2000 g);5. Reducing the rotor speed to 60 rpm for the duration of themeasurement (1-2 min), each tube being sampled once every second; and6. Generating a time series of photon counts for each sample for displayand/or input to a computer algorithm for evaluation.

An example of the data from a 16-channel measurement, seen in FIG. 40,shows an LOD comparable to that of the single tube method. While this 16channel sensor will measure 16 samples as designed, larger samplenumbers are possible by increasing the number of channels, thoughphysical size and the statistics of sampling will ultimately dictatepractical limits. Similarly, smaller sample numbers are possible bydecreasing either the number of samples loaded onto a sensor, or byreducing the number of channels on the sensor. A CAD drawing of the16-channel portable sensor design is shown in FIG. 41.

A further implementation of this 16-channel design is referred to as aTCAN sensor. The TCAN (Triggered-CANARY) biosensor is an automatedbiosensor which combines both aerosol collection and B-cell liquiddelivery into an integrated radial disc format. The TCAN CANARY disc(CD) (FIG. 42) interfaces with a manifold assembly which splits an airflow into separate channels. The aerosol collection assembly (FIG. 43)uses dry impaction techniques to then localize particles from the airflow into the bottom of clear plastic tubes.

After impaction of aerosol particles, the CD interfaces with themanifold assembly to actuate valves located in the disc. The disc israpidly spun, which in turn causes the emittor cell liquid to deliver toindividual tubes using centrifugal force (FIG. 44). An optical detectoris then used to identify potential bioagents based on the photon outputemittor cells interacting with the aerosol particles. This process ofaerosol collection and emittor cell delivery can be repeated severaltimes in one disc. This feature allows multiple CANARY assays to beperformed after several trigger events without changing the CD.

Toxin Detection Example

Detection of soluble proteins can be achieved using a variety ofmethods. For example, in one method, two antibodies can be expressed inthe same emittor cell, wherein the two antibodies are each against adifferent epitope on the same molecule. The antibodies are thencrosslinked by monomeric antigen (FIG. 48). It should be pointed outthat the sorting of antibodies in the secretory pathway is idealized inthe schematic of FIG. 48. In one example, the antibodies can beheterofunctional, i.e., one antibody can have two different functionalantigen binding sites. In another example, each antibody has only onefunctional antigen binding site. This method depends on two factors: (1)multiple functional antibodies are expressed by the same emittor celland (2) two, linked epitopes are sufficient to stimulate emittor cells(although more than one of these pairs may be required to stimulate agiven cell).

In one experiment, multiple, functional antibodies were expressed in thesame emittor cell line (FIG. 49). A single cell line expressingantibodies against Bacillus anthracis and Yersinia pestis was generated.This clonal cell line reacts against both antigens with goodsensitivity. It will be understood that two antibodies against twoepitopes on the same soluble monomer can also be functionally expressed.Furthermore, two linked epitopes is sufficient to stimulate emittorcells.

A second method for detecting soluble, monomeric antigens is tocrosslink the soluble antigen to make it appear multivalent to theemittor cell (FIG. 50). This crosslinking can be done by attaching theprotein to beads, either via tags, in the case of recombinant proteins,or via antibody, as has been demonstrated for botulinum toxin Hcfragment. There are a variety of other possible methods for effectivelycrosslinking the antigen, as will be understood by those of skill in theart, including precipitation of antigen with trichloroacetic acid (TCA),heat, or ethanol, and attachment of the antigen to a solid phase vialigands, antibodies, or chemical functional groups. This crosslinkedmonomer can then be detected using emittor cells expressing antibodythat recognizes an epitope still available on the crosslinked antigen.

This second method has been demonstrated in practice, using the heavychain of botulinum toxin type A (BoNT/A Hc) as the soluble, monomerictarget protein (FIG. 51) and antibodies described in Pless et al.,Infection and Immunity (2001) 570-574. Monoclonal antibody (6E10-10)against one epitope was crosslinked to protein G-coated beads. Thesebeads were incubated with BoNT/A Hc for 3 hrs at 4° C., washed, and usedto stimulate emittor cells expressing a second antibody (6B2-2) thatrecognizes a different BoNT/A Hc epitope. The BoNT/A Hc-decorated beadseffectively stimulated the emittor cells, with an LOD of about 6 ng.Emittor cells expressing the same antibody as that used to bind theBoNT/A to the beads were not stimulated, indicating that the emittorreaction was not caused by aggregation of the target protein.

Chemical Detection Example

Chemical detection is of importance in both military and clinicalsettings. It is possible that some chemicals may have two epitopes towhich antibodies can bind independently. In such cases the methods forchemical detection would be identical to that for toxins detectionoutlined above. In many cases, however, there will not be twoindependent epitopes on the chemical of interest. In such cases it willbe necessary to modify the chemical such that it is capable ofstimulating the emittor cell. Four of these modifications are outlinedbelow.

1. Immobilize the chemical of interest on a solid support. Generateemittor cells expressing antibodies that recognize the portion of thechemical that remains available. When the density of the immobilizedchemical on the solid support is high enough, antibodies on the emittorcell surface will be immobilized close enough to each other to stimulatethe cell. This is analogous with the scheme for toxin detection shown inFIG. 50.2. First, generate peptide(s) that bind specifically to the chemical.Next, generate antibodies that bind specifically to the chemical-peptidecomplex. If the chemical-peptide complex is composed of two or moreepitopes, the complex can be detected by either of the two-antibodytechniques outlined in the section on toxin detection. If the complex isonly composed of one specific epitope, then an additional epitope, suchas digoxigenin, can be added synthetically to the peptide (FIG. 52) Thecomplex would then contain two antibody binding sites: (1) the epitopeformed by the peptide-chemical complex and (2) the digoxigenin epitope.Only in the presence of chemical would both epitopes be present. Thesetwo epitopes can then be detected by either of the two-antibodytechniques outlined in the section on toxin detection.3. Generate two peptides that specifically bind to the chemical (or toeach other in the presence of the chemical). Each of these peptides canbe synthetically tagged, such that only in the presence of chemicalwould two epitopes be bound to each other, and therefore detectable bythe emittor cell (FIG. 53). Alternatively, one or more antibodies can bemade against the peptide-chemical complex, and the presence of chemicaldetected as above using a combination of antibodies against the complex,or one antibody against the complex and one antibody against a peptidetag.4. As above, generate peptide(s) that bind specifically to the chemical,and generate antibodies that specifically bind to the peptide-chemicalcomplex. Dimerize the chemical-binding peptide, so that if the dimerbinds to two chemicals, it will contain two antibody binding sites. Thiscomplex can be detected by emittor cells expressing an antibody againstthe chemical-peptide complex.

Peptides that bind to small molecules have been isolated fromcombinatorial libraries. These molecules include porphyrin (Nakamura etal., Biosensors and Bioelectronics 2001, 16: 1095-1100) tryptophan(Sugimoto et al., 1999, 677-678) and cadmium (Mejare et al., 1998,Protein Engineering 11(6): 489-494). However, the use of proteins in theplace of peptides may yield higher affinity binders. Libraries have beenconstructed in which the binding sites have been combinatoriallydefined, and these can be used to isolate those binding to smallmolecules. Such a library using lipocalin as the starting protein hasbeen used to isolate binders to digoxigenin variants (Schlehuber andSkerra, 2002, Biophysical Chemistry 96: 213-228). This approach can beused starting with any number of other proteins, but particularly thosethat might be expected to already have some binding activity with thechemical target (for example, acetylcholinesterase, in the case of VXand Sarin).

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

The relevant teachings of all the references, patents and patentapplications cited herein are incorporated herein by reference in theirentirety.

1-15. (canceled)
 16. A method of detecting a soluble antigen in a samplecomprising the steps of: a) crosslinking the soluble antigen, therebyproducing a crosslinked antigen; b) combining the crosslinked antigenwith an emittor cell comprising an antibody that binds to thecrosslinked antigen, wherein binding of the antibody to the crosslinkedantigen results in an increase in intracellular calcium, and whereinsaid emittor cell further comprises an emittor molecule that emits aphoton in response to the increase in intracellular calcium; and c)measuring photon emission from the emittor cell, thereby detecting asoluble antigen in a sample.
 17. A method of detecting a soluble antigenin a sample comprising the steps of: a) crosslinking the soluble antigento a solid substrate, thereby producing a crosslinked soluble antigenbound to a solid substrate; b) adding an emittor cell to the crosslinkedsoluble antigen bound to the solid substrate, wherein said emittor cellcomprises an antibody that binds an epitope on the soluble antigen,wherein binding of the antibody to the crosslinked soluble antigen boundto the solid support results in an increase in intracellular calcium,and wherein said emittor cell further comprises an emittor molecule thatemits a photon in response to the increase in intracellular calcium; andc) measuring photon emission from the emittor cell, thereby detecting asoluble antigen in a sample.
 18. The method of claim 17, wherein thesoluble antigen is a protein.
 19. The method of claim 17, wherein thesoluble antigen is a chemical. 20-26. (canceled)
 27. The method of claim16, wherein, the antibody that binds to the crosslinked antigen isselected from the group consisting of a chimeric antibody, asingle-chain antibody, a monoclonal antibody and a polyclonal antibody.28. The method of claim 16, wherein, the soluble antigen is selectedfrom the group consisting of a protein and a chemical.
 29. The method ofclaim 16, wherein the soluble antigen is crosslinked using one or moreagents selected from the group consisting of molecules, antibodies,chemical compounds and ligands.
 30. The method of claim 29, wherein theone or more agents crosslink the soluble antigen by precipitation of thesoluble antigen or by attachment of the soluble antigen to a solidphase.
 31. The method of claim 16, wherein the emittor molecule is acalcium-sensitive luminescent molecule or a calcium-sensitivefluorescent molecule.
 32. The method of claim 31, wherein the emittormolecule is selected from the group consisting of aequorin, obelin,thalassicolin, mitrocomin, clytin, mnemopsin, berovin, Indo-1, Fura-2,Quin-2, Fluo-3, Rhod-2, calcium green, BAPTA, a cameleon andcombinations thereof.
 33. The method of claim 16, wherein the emittorcell is a cell selected from the group consisting of a prokaryotic cell,a eukaryotic cell and a non-living cell.
 34. The method of claim 33,wherein the emittor cell is selected from the group consisting of aB-cell, a T-cell, a macrophage cell, a mast cell and a fibroblast. 35.The method of claim 17, wherein the soluble antigen is crosslinked tothe solid substrate using one or more agents selected from the groupconsisting of molecules, antibodies, chemical compounds and ligands. 36.The method of claim 17, wherein the solid substrate comprises one ormore substrates selected from the group consisting of a multi-wellplate, a microcentrifuge tube, a filter unit, a bead, a cell, a chargedmolecule and a bacterium.
 37. The method of claim 17, wherein, theantibody that binds an epitope on the soluble antigen is selected fromthe group consisting of a chimeric antibody, a single-chain antibody, amonoclonal antibody and a polyclonal antibody.
 38. The method of claim17, wherein the emittor molecule comprises a calcium-sensitiveluminescent molecule or a calcium-sensitive fluorescent molecule. 39.The method of claim 38, wherein the emittor molecule is selected fromthe group consisting of aequorin, obelin, thalassicolin, mitrocomin(halistaurin), clytin (phialidin), mnemopsin, berovin, Indo-1, Fura-2,Quin-2, Fluo-3, Rhod-2, calcium green, BAPTA, a cameleon andcombinations thereof.
 40. The method of claim 17, wherein the emittorcell comprises a cell selected from the group consisting of aprokaryotic cell, a eukaryotic cell and a non-living cell.
 41. Themethod of claim 40, wherein the emittor cell is selected from the groupconsisting of a B-cell, a T-cell, a macrophage cell, a mast cell and afibroblast.